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Sample records for meiotic recombination protein

  1. Recombination Proteins Mediate Meiotic Spatial Chromosome Organization and Pairing

    Science.gov (United States)

    Storlazzi, Aurora; Gargano, Silvana; Ruprich-Robert, Gwenael; Falque, Matthieu; David, Michelle; Kleckner, Nancy; Zickler, Denise

    2010-01-01

    SUMMARY Meiotic chromosome pairing involves not only recognition of homology but also juxtaposition of entire chromosomes in a topologically regular way. Analysis of filamentous fungus Sordaria macrospora reveals that recombination proteins Mer3, Msh4 and Mlh1 play direct roles in all of these aspects, in advance of their known roles in recombination. Absence of Mer3 helicase results in interwoven chromosomes, thereby revealing the existence of features that specifically ensure “entanglement avoidance”. Entanglements that remain at zygotene, i.e. “interlockings”, require Mlh1 for resolution, likely to eliminate constraining recombinational connections. Patterns of Mer3 and Msh4 foci along aligned chromosomes show that the double-strand breaks mediating homologous alignment have spatially separated ends, one localized to each partner axis, and that pairing involves interference among developing interhomolog interactions. We propose that Mer3, Msh4 and Mlh1 execute all of these roles during pairing by modulating the state of nascent double-strand break/partner DNA contacts within axis-associated recombination complexes. PMID:20371348

  2. Regulation of Meiotic Recombination

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    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

  3. Meiotic recombination in human oocytes.

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    Edith Y Cheng

    2009-09-01

    Full Text Available Studies of human trisomies indicate a remarkable relationship between abnormal meiotic recombination and subsequent nondisjunction at maternal meiosis I or II. Specifically, failure to recombine or recombination events located either too near to or too far from the centromere have been linked to the origin of human trisomies. It should be possible to identify these abnormal crossover configurations by using immunofluorescence methodology to directly examine the meiotic recombination process in the human female. Accordingly, we initiated studies of crossover-associated proteins (e.g., MLH1 in human fetal oocytes to analyze their number and distribution on nondisjunction-prone human chromosomes and, more generally, to characterize genome-wide levels of recombination in the human female. Our analyses indicate that the number of MLH1 foci is lower than predicted from genetic linkage analysis, but its localization pattern conforms to that expected for a crossover-associated protein. In studies of individual chromosomes, our observations provide evidence for the presence of "vulnerable" crossover configurations in the fetal oocyte, consistent with the idea that these are subsequently translated into nondisjunctional events in the adult oocyte.

  4. Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing.

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    Olivier J Becherel

    2013-04-01

    Full Text Available Senataxin, mutated in the human genetic disorder ataxia with oculomotor apraxia type 2 (AOA2, plays an important role in maintaining genome integrity by coordination of transcription, DNA replication, and the DNA damage response. We demonstrate that senataxin is essential for spermatogenesis and that it functions at two stages in meiosis during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI. Disruption of the Setx gene caused persistence of DNA double-strand breaks, a defect in disassembly of Rad51 filaments, accumulation of DNA:RNA hybrids (R-loops, and ultimately a failure of crossing-over. Senataxin localised to the XY body in a Brca1-dependent manner, and in its absence there was incomplete localisation of DNA damage response proteins to the XY chromosomes and ATR was retained on the axial elements of these chromosomes, failing to diffuse out into chromatin. Furthermore persistence of RNA polymerase II activity, altered ubH2A distribution, and abnormal XY-linked gene expression in Setx⁻/⁻ revealed an essential role for senataxin in MSCI. These data support key roles for senataxin in coordinating meiotic crossing-over with transcription and in gene silencing to protect the integrity of the genome.

  5. Initiation of Meiotic Recombination in Mammals

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    Rajeev Kumar

    2010-12-01

    Full Text Available Meiotic recombination is initiated by the induction of programmed DNA double strand breaks (DSBs. DSB repair promotes homologous interactions and pairing and leads to the formation of crossovers (COs, which are required for the proper reductional segregation at the first meiotic division. In mammals, several hundred DSBs are generated at the beginning of meiotic prophase by the catalytic activity of SPO11. Currently it is not well understood how the frequency and timing of DSB formation and their localization are regulated. Several approaches in humans and mice have provided an extensive description of the localization of initiation events based on CO mapping, leading to the identification and characterization of preferred sites (hotspots of initiation. This review presents the current knowledge about the proteins known to be involved in this process, the sites where initiation takes place, and the factors that control hotspot localization.

  6. RPA homologs and ssDNA processing during meiotic recombination.

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    Ribeiro, Jonathan; Abby, Emilie; Livera, Gabriel; Martini, Emmanuelle

    2016-06-01

    Meiotic homologous recombination is a specialized process that involves homologous chromosome pairing and strand exchange to guarantee proper chromosome segregation and genetic diversity. The formation and repair of DNA double-strand breaks (DSBs) during meiotic recombination differs from those during mitotic recombination in that the homologous chromosome rather than the sister chromatid is the preferred repair template. The processing of single-stranded DNA (ssDNA) formed on intermediate recombination structures is central to driving the specific outcomes of DSB repair during meiosis. Replication protein A (RPA) is the main ssDNA-binding protein complex involved in DNA metabolism. However, the existence of RPA orthologs in plants and the recent discovery of meiosis specific with OB domains (MEIOB), a widely conserved meiosis-specific RPA1 paralog, strongly suggest that multiple RPA complexes evolved and specialized to subdivide their roles during DNA metabolism. Here we review ssDNA formation and maturation during mitotic and meiotic recombination underlying the meiotic specific features. We describe and discuss the existence and properties of MEIOB and multiple RPA subunits in plants and highlight how they can provide meiosis-specific fates to ssDNA processing during homologous recombination. Understanding the functions of these RPA homologs and how they interact with the canonical RPA subunits is of major interest in the fields of meiosis and DNA repair.

  7. Polyploidization increases meiotic recombination frequency in Arabidopsis

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    Rehmsmeier Marc

    2011-04-01

    Full Text Available Abstract Background Polyploidization is the multiplication of the whole chromosome complement and has occurred frequently in vascular plants. Maintenance of stable polyploid state over generations requires special mechanisms to control pairing and distribution of more than two homologous chromosomes during meiosis. Since a minimal number of crossover events is essential for correct chromosome segregation, we investigated whether polyploidy has an influence on the frequency of meiotic recombination. Results Using two genetically linked transgenes providing seed-specific fluorescence, we compared a high number of progeny from diploid and tetraploid Arabidopsis plants. We show that rates of meiotic recombination in reciprocal crosses of genetically identical diploid and autotetraploid Arabidopsis plants were significantly higher in tetraploids compared to diploids. Although male and female gametogenesis differ substantially in meiotic recombination frequency, both rates were equally increased in tetraploids. To investigate whether multivalent formation in autotetraploids was responsible for the increased recombination rates, we also performed corresponding experiments with allotetraploid plants showing strict bivalent pairing. We found similarly increased rates in auto- and allotetraploids, suggesting that the ploidy effect is independent of chromosome pairing configurations. Conclusions The evolutionary success of polyploid plants in nature and under domestication has been attributed to buffering of mutations and sub- and neo-functionalization of duplicated genes. Should the data described here be representative for polyploid plants, enhanced meiotic recombination, and the resulting rapid creation of genetic diversity, could have also contributed to their prevalence.

  8. Meiotic recombination hotspots - a comparative view.

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    Choi, Kyuha; Henderson, Ian R

    2015-07-01

    During meiosis homologous chromosomes pair and undergo reciprocal genetic exchange, termed crossover. Meiotic recombination has a profound effect on patterns of genetic variation and is an important tool during crop breeding. Crossovers initiate from programmed DNA double-stranded breaks that are processed to form single-stranded DNA, which can invade a homologous chromosome. Strand invasion events mature into double Holliday junctions that can be resolved as crossovers. Extensive variation in the frequency of meiotic recombination occurs along chromosomes and is typically focused in narrow hotspots, observed both at the level of DNA breaks and final crossovers. We review methodologies to profile hotspots at different steps of the meiotic recombination pathway that have been used in different eukaryote species. We then discuss what these studies have revealed concerning specification of hotspot locations and activity and the contributions of both genetic and epigenetic factors. Understanding hotspots is important for interpreting patterns of genetic variation in populations and how eukaryotic genomes evolve. In addition, manipulation of hotspots will allow us to accelerate crop breeding, where meiotic recombination distributions can be limiting. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

  9. RNAi and heterochromatin repress centromeric meiotic recombination

    DEFF Research Database (Denmark)

    Ellermeier, Chad; Higuchi, Emily C; Phadnis, Naina

    2010-01-01

    During meiosis, the formation of viable haploid gametes from diploid precursors requires that each homologous chromosome pair be properly segregated to produce an exact haploid set of chromosomes. Genetic recombination, which provides a physical connection between homologous chromosomes, is essen......During meiosis, the formation of viable haploid gametes from diploid precursors requires that each homologous chromosome pair be properly segregated to produce an exact haploid set of chromosomes. Genetic recombination, which provides a physical connection between homologous chromosomes....... Surprisingly, one mutant derepressed for recombination in the heterochromatic mating-type region during meiosis and several mutants derepressed for centromeric gene expression during mitotic growth are not derepressed for centromeric recombination during meiosis. These results reveal a complex relation between...... types of repression by heterochromatin. Our results also reveal a previously undemonstrated role for RNAi and heterochromatin in the repression of meiotic centromeric recombination and, potentially, in the prevention of birth defects by maintenance of proper chromosome segregation during meiosis....

  10. Finding trans-regulatory genes and protein complexes modulating meiotic recombination hotspots of human, mouse and yeast.

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    Wu, Min; Kwoh, Chee-Keong; Li, Xiaoli; Zheng, Jie

    2014-09-11

    The regulatory mechanism of recombination is one of the most fundamental problems in genomics, with wide applications in genome wide association studies (GWAS), birth-defect diseases, molecular evolution, cancer research, etc. Recombination events cluster into short genomic regions called "recombination hotspots". Recently, a zinc finger protein PRDM9 was reported to regulate recombination hotspots in human and mouse genomes. In addition, a 13-mer motif contained in the binding sites of PRDM9 is found to be enriched in human hotspots. However, this 13-mer motif only covers a fraction of hotspots, indicating that PRDM9 is not the only regulator of recombination hotspots. Therefore, the challenge of discovering other regulators of recombination hotspots becomes significant. Furthermore, recombination is a complex process. Hence, multiple proteins acting as machinery, rather than individual proteins, are more likely to carry out this process in a precise and stable manner. Therefore, the extension of the prediction of individual trans-regulators to protein complexes is also highly desired. In this paper, we introduce a pipeline to identify genes and protein complexes associated with recombination hotspots. First, we prioritize proteins associated with hotspots based on their preference of binding to hotspots and coldspots. Second, using the above identified genes as seeds, we apply the Random Walk with Restart algorithm (RWR) to propagate their influences to other proteins in protein-protein interaction (PPI) networks. Hence, many proteins without DNA-binding information will also be assigned a score to implicate their roles in recombination hotspots. Third, we construct sub-PPI networks induced by top genes ranked by RWR for various species (e.g., yeast, human and mouse) and detect protein complexes in those sub-PPI networks. The GO term analysis show that our prioritizing methods and the RWR algorithm are capable of identifying novel genes associated with

  11. Prevention of DNA Rereplication Through a Meiotic Recombination Checkpoint Response

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    Nicole A. Najor

    2016-12-01

    Full Text Available In the budding yeast Saccharomyces cerevisiae, unnatural stabilization of the cyclin-dependent kinase inhibitor Sic1 during meiosis can trigger extra rounds of DNA replication. When programmed DNA double-strand breaks (DSBs are generated but not repaired due to absence of DMC1, a pathway involving the checkpoint gene RAD17 prevents this DNA rereplication. Further genetic analysis has now revealed that prevention of DNA rereplication also requires MEC1, which encodes a protein kinase that serves as a central checkpoint regulator in several pathways including the meiotic recombination checkpoint response. Downstream of MEC1, MEK1 is required through its function to inhibit repair between sister chromatids. By contrast, meiotic recombination checkpoint effectors that regulate gene expression and cyclin-dependent kinase activity are not necessary. Phosphorylation of histone H2A, which is catalyzed by Mec1 and the related Tel1 protein kinase in response to DSBs, and can help coordinate activation of the Rad53 checkpoint protein kinase in the mitotic cell cycle, is required for the full checkpoint response. Phosphorylation sites that are targeted by Rad53 in a mitotic S phase checkpoint response are also involved, based on the behavior of cells containing mutations in the DBF4 and SLD3 DNA replication genes. However, RAD53 does not appear to be required, nor does RAD9, which encodes a mediator of Rad53, consistent with their lack of function in the recombination checkpoint pathway that prevents meiotic progression. While this response is similar to a checkpoint mechanism that inhibits initiation of DNA replication in the mitotic cell cycle, the evidence points to a new variation on DNA replication control.

  12. Hybrid Sterility Locus on Chromosome X Controls Meiotic Recombination Rate in Mouse.

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    Maria Balcova

    2016-04-01

    Full Text Available Meiotic recombination safeguards proper segregation of homologous chromosomes into gametes, affects genetic variation within species, and contributes to meiotic chromosome recognition, pairing and synapsis. The Prdm9 gene has a dual role, it controls meiotic recombination by determining the genomic position of crossover hotspots and, in infertile hybrids of house mouse subspecies Mus m. musculus (Mmm and Mus m. domesticus (Mmd, it further functions as the major hybrid sterility gene. In the latter role Prdm9 interacts with the hybrid sterility X 2 (Hstx2 genomic locus on Chromosome X (Chr X by a still unknown mechanism. Here we investigated the meiotic recombination rate at the genome-wide level and its possible relation to hybrid sterility. Using immunofluorescence microscopy we quantified the foci of MLH1 DNA mismatch repair protein, the cytological counterparts of reciprocal crossovers, in a panel of inter-subspecific chromosome substitution strains. Two autosomes, Chr 7 and Chr 11, significantly modified the meiotic recombination rate, yet the strongest modifier, designated meiotic recombination 1, Meir1, emerged in the 4.7 Mb Hstx2 genomic locus on Chr X. The male-limited transgressive effect of Meir1 on recombination rate parallels the male-limited transgressive role of Hstx2 in hybrid male sterility. Thus, both genetic factors, the Prdm9 gene and the Hstx2/Meir1 genomic locus, indicate a link between meiotic recombination and hybrid sterility. A strong female-specific modifier of meiotic recombination rate with the effect opposite to Meir1 was localized on Chr X, distally to Meir1. Mapping Meir1 to a narrow candidate interval on Chr X is an important first step towards positional cloning of the respective gene(s responsible for variation in the global recombination rate between closely related mouse subspecies.

  13. Hybrid Sterility Locus on Chromosome X Controls Meiotic Recombination Rate in Mouse.

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    Balcova, Maria; Faltusova, Barbora; Gergelits, Vaclav; Bhattacharyya, Tanmoy; Mihola, Ondrej; Trachtulec, Zdenek; Knopf, Corinna; Fotopulosova, Vladana; Chvatalova, Irena; Gregorova, Sona; Forejt, Jiri

    2016-04-01

    Meiotic recombination safeguards proper segregation of homologous chromosomes into gametes, affects genetic variation within species, and contributes to meiotic chromosome recognition, pairing and synapsis. The Prdm9 gene has a dual role, it controls meiotic recombination by determining the genomic position of crossover hotspots and, in infertile hybrids of house mouse subspecies Mus m. musculus (Mmm) and Mus m. domesticus (Mmd), it further functions as the major hybrid sterility gene. In the latter role Prdm9 interacts with the hybrid sterility X 2 (Hstx2) genomic locus on Chromosome X (Chr X) by a still unknown mechanism. Here we investigated the meiotic recombination rate at the genome-wide level and its possible relation to hybrid sterility. Using immunofluorescence microscopy we quantified the foci of MLH1 DNA mismatch repair protein, the cytological counterparts of reciprocal crossovers, in a panel of inter-subspecific chromosome substitution strains. Two autosomes, Chr 7 and Chr 11, significantly modified the meiotic recombination rate, yet the strongest modifier, designated meiotic recombination 1, Meir1, emerged in the 4.7 Mb Hstx2 genomic locus on Chr X. The male-limited transgressive effect of Meir1 on recombination rate parallels the male-limited transgressive role of Hstx2 in hybrid male sterility. Thus, both genetic factors, the Prdm9 gene and the Hstx2/Meir1 genomic locus, indicate a link between meiotic recombination and hybrid sterility. A strong female-specific modifier of meiotic recombination rate with the effect opposite to Meir1 was localized on Chr X, distally to Meir1. Mapping Meir1 to a narrow candidate interval on Chr X is an important first step towards positional cloning of the respective gene(s) responsible for variation in the global recombination rate between closely related mouse subspecies.

  14. Absence of SUN-domain protein Slp1 blocks karyogamy and switches meiotic recombination and synapsis from homologs to sister chromatids

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    Vasnier, Christelle; de Muyt, Arnaud; Zhang, Liangran; Tessé, Sophie; Kleckner, Nancy E.; Zickler, Denise; Espagne, Eric

    2014-01-01

    Karyogamy, the process of nuclear fusion is required for two haploid gamete nuclei to form a zygote. Also, in haplobiontic organisms, karyogamy is required to produce the diploid nucleus/cell that then enters meiosis. We identify sun like protein 1 (Slp1), member of the mid–Sad1p, UNC-84–domain ubiquitous family, as essential for karyogamy in the filamentous fungus Sordaria macrospora, thus uncovering a new function for this protein family. Slp1 is required at the last step, nuclear fusion, not for earlier events including nuclear movements, recognition, and juxtaposition. Correspondingly, like other family members, Slp1 localizes to the endoplasmic reticulum and also to its extensions comprising the nuclear envelope. Remarkably, despite the absence of nuclear fusion in the slp1 null mutant, meiosis proceeds efficiently in the two haploid “twin” nuclei, by the same program and timing as in diploid nuclei with a single dramatic exception: the normal prophase program of recombination and synapsis between homologous chromosomes, including loading of recombination and synaptonemal complex proteins, occurs instead between sister chromatids. Moreover, the numbers of recombination-initiating double-strand breaks (DSBs) and ensuing recombinational interactions, including foci of the essential crossover factor Homo sapiens enhancer of invasion 10 (Hei10), occur at half the diploid level in each haploid nucleus, implying per-chromosome specification of DSB formation. Further, the distribution of Hei10 foci shows interference like in diploid meiosis. Centromere and spindle dynamics, however, still occur in the diploid mode during the two meiotic divisions. These observations imply that the prophase program senses absence of karyogamy and/or absence of a homolog partner and adjusts the interchromosomal interaction program accordingly. PMID:25210014

  15. MEIOB targets single-strand DNA and is necessary for meiotic recombination.

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    Benoit Souquet

    Full Text Available Meiotic recombination is a mandatory process for sexual reproduction. We identified a protein specifically implicated in meiotic homologous recombination that we named: meiosis specific with OB domain (MEIOB. This protein is conserved among metazoan species and contains single-strand DNA binding sites similar to those of RPA1. Our studies in vitro revealed that both recombinant and endogenous MEIOB can be retained on single-strand DNA. Those in vivo demonstrated the specific expression of Meiob in early meiotic germ cells and the co-localization of MEIOB protein with RPA on chromosome axes. MEIOB localization in Dmc1 (-/- spermatocytes indicated that it accumulates on resected DNA. Homologous Meiob deletion in mice caused infertility in both sexes, due to a meiotic arrest at a zygotene/pachytene-like stage. DNA double strand break repair and homologous chromosome synapsis were impaired in Meiob (-/- meiocytes. Interestingly MEIOB appeared to be dispensable for the initial loading of recombinases but was required to maintain a proper number of RAD51 and DMC1 foci beyond the zygotene stage. In light of these findings, we propose that RPA and this new single-strand DNA binding protein MEIOB, are essential to ensure the proper stabilization of recombinases which is required for successful homology search and meiotic recombination.

  16. Meiotic recombination, synapsis, meiotic inactivation and sperm aneuploidy in a chromosome 1 inversion carrier.

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    Kirkpatrick, Gordon; Chow, Victor; Ma, Sai

    2012-01-01

    Disrupted meiotic behaviour of inversion carriers may be responsible for suboptimal sperm parameters in these carriers. This study investigated meiotic recombination, synapsis, transcriptional silencing and chromosome segregation effects in a pericentric inv(1) carrier. Recombination (MLH1), synapsis (SYCP1, SYCP3) and transcriptional inactivation (γH2AX, BRCA1) were examined by fluorescence immunostaining. Chromosome specific rates of recombination were determined by fluorescence in-situ hybridization. Furthermore, testicular sperm was examined for aneuploidy and segregation of the inv(1). Our findings showed that global recombination rates were similar to controls. Recombination on the inv(1) and the sex chromosomes were reduced. The inv(1) associated with the XY body in 43.4% of cells, in which XY recombination was disproportionately absent, and 94.3% of cells displayed asynapsed regions which displayed meiotic silencing regardless of their association with the XY body. Furthermore, a low frequency of chromosomal imbalance was observed in spermatozoa (3.4%). Our results suggest that certain inversion carriers may display unimpaired global recombination and impaired recombination on the involved and the sex chromosomes during meiosis. Asynapsis or inversion-loop formation in the inverted region may be responsible for impaired spermatogenesis and may prevent sperm-chromosome imbalance. Copyright © 2011 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  17. Meiotic recombination breakpoints are associated with open chromatin and enriched with repetitive DNA elements in potato

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    Meiotic recombination provides the framework for the genetic variation in natural and artificial populations of eukaryotes through the creation of novel haplotypes. Thus, determining the molecular characteristics of meiotic recombination remains essential for future plant breeding efforts, which hea...

  18. Protein Determinants of Meiotic DNA Break Hotspots

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    Fowler, Kyle R.; Gutiérrez-Velasco, Susana

    2013-01-01

    SUMMARY Meiotic recombination, crucial for proper chromosome segregation and genome evolution, is initiated by programmed DNA double-strand breaks (DSBs) in yeasts and likely all sexually reproducing species. In fission yeast, DSBs occur up to hundreds of times more frequently at special sites, called hotspots, than in other regions of the genome. What distinguishes hotspots from cold regions is an unsolved problem, although transcription factors determine some hotspots. We report the discovery that three coiled-coil proteins – Rec25, Rec27, and Mug20 – bind essentially all hotspots with unprecedented specificity even without DSB formation. These small proteins are components of linear elements, are related to synaptonemal complex proteins, and are essential for nearly all DSBs at most hotspots. Our results indicate these hotspot determinants activate or stabilize the DSB-forming protein Rec12 (Spo11 homolog) rather than promote its binding to hotspots. We propose a new paradigm for hotspot determination and crossover control by linear element proteins. PMID:23395004

  19. Genetic analysis of variation in human meiotic recombination.

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    Reshmi Chowdhury

    2009-09-01

    Full Text Available The number of recombination events per meiosis varies extensively among individuals. This recombination phenotype differs between female and male, and also among individuals of each gender. In this study, we used high-density SNP genotypes of over 2,300 individuals and their offspring in two datasets to characterize recombination landscape and to map the genetic variants that contribute to variation in recombination phenotypes. We found six genetic loci that are associated with recombination phenotypes. Two of these (RNF212 and an inversion on chromosome 17q21.31 were previously reported in the Icelandic population, and this is the first replication in any other population. Of the four newly identified loci (KIAA1462, PDZK1, UGCG, NUB1, results from expression studies provide support for their roles in meiosis. Each of the variants that we identified explains only a small fraction of the individual variation in recombination. Notably, we found different sequence variants associated with female and male recombination phenotypes, suggesting that they are regulated by different genes. Characterization of genetic variants that influence natural variation in meiotic recombination will lead to a better understanding of normal meiotic events as well as of non-disjunction, the primary cause of pregnancy loss.

  20. Managing meiotic recombination in plant breeding

    NARCIS (Netherlands)

    Wijnker, T.G.; Jong, de J.H.S.G.M.

    2008-01-01

    Crossover recombination is a crucial process in plant breeding because it allows plant breeders to create novel allele combnations on chromosomes that can be used for breeding superior F1 hybrids. Gaining control over this process, in terms of increasing crossover incidence, altering crossover

  1. ARG-walker: inference of individual specific strengths of meiotic recombination hotspots by population genomics analysis.

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    Chen, Hao; Yang, Peng; Guo, Jing; Kwoh, Chee Keong; Przytycka, Teresa M; Zheng, Jie

    2015-01-01

    Meiotic recombination hotspots play important roles in various aspects of genomics, but the underlying mechanisms for regulating the locations and strengths of recombination hotspots are not yet fully revealed. Most existing algorithms for estimating recombination rates from sequence polymorphism data can only output average recombination rates of a population, although there is evidence for the heterogeneity in recombination rates among individuals. For genome-wide association studies (GWAS) of recombination hotspots, an efficient algorithm that estimates the individualized strengths of recombination hotspots is highly desirable. In this work, we propose a novel graph mining algorithm named ARG-walker, based on random walks on ancestral recombination graphs (ARG), to estimate individual-specific recombination hotspot strengths. Extensive simulations demonstrate that ARG-walker is able to distinguish the hot allele of a recombination hotspot from the cold allele. Integrated with output of ARG-walker, we performed GWAS on the phased haplotype data of the 22 autosome chromosomes of the HapMap Asian population samples of Chinese and Japanese (JPT+CHB). Significant cis-regulatory signals have been detected, which is corroborated by the enrichment of the well-known 13-mer motif CCNCCNTNNCCNC of PRDM9 protein. Moreover, two new DNA motifs have been identified in the flanking regions of the significantly associated SNPs (single nucleotide polymorphisms), which are likely to be new cis-regulatory elements of meiotic recombination hotspots of the human genome. Our results on both simulated and real data suggest that ARG-walker is a promising new method for estimating the individual recombination variations. In the future, it could be used to uncover the mechanisms of recombination regulation and human diseases related with recombination hotspots.

  2. Fine-scale variation in meiotic recombination in Mimulus inferred from population shotgun sequencing

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    Hellsten, Uffe [USDOE Joint Genome Inst., Walnut Creek, CA (United States); Wright, Kevin M. [Harvard Univ., Cambridge, MA (United States); Jenkins, Jerry [USDOE Joint Genome Inst., Walnut Creek, CA (United States); HudsonAlpha Inst. of Biotechnology, Huntsville, AL (United States); Shu, Shengqiang [USDOE Joint Genome Inst., Walnut Creek, CA (United States); Yuan, Yao-Wu [Univ. of Connecticut, Storrs, CT (United States); Wessler, Susan R. [Univ. of California, Riverside, CA (United States); Schmutz, Jeremy [USDOE Joint Genome Inst., Walnut Creek, CA (United States); HudsonAlpha Inst. of Biotechnology, Huntsville, AL (United States); Willis, John H. [Duke Univ., Durham, NC (United States); Rokhsar, Daniel S. [USDOE Joint Genome Inst., Walnut Creek, CA (United States); Univ. of California, Berkeley, CA (United States)

    2013-11-13

    Meiotic recombination rates can vary widely across genomes, with hotspots of intense activity interspersed among cold regions. In yeast, hotspots tend to occur in promoter regions of genes, whereas in humans and mice hotspots are largely defined by binding sites of the PRDM9 protein. To investigate the detailed recombination pattern in a flowering plant we use shotgun resequencing of a wild population of the monkeyflower Mimulus guttatus to precisely locate over 400,000 boundaries of historic crossovers or gene conversion tracts. Their distribution defines some 13,000 hotspots of varying strengths, interspersed with cold regions of undetectably low recombination. Average recombination rates peak near starts of genes and fall off sharply, exhibiting polarity. Within genes, recombination tracts are more likely to terminate in exons than in introns. The general pattern is similar to that observed in yeast, as well as in PRDM9-knockout mice, suggesting that recombination initiation described here in Mimulus may reflect ancient and conserved eukaryotic mechanisms

  3. A new seed-based assay for meiotic recombination in Arabidopsis thaliana.

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    Melamed-Bessudo, C.; Yehuda, E.; Stuitje, A.R.; Levy, A.A.

    2005-01-01

    Meiotic recombination is a fundamental biological process that plays a central role in the evolution and breeding of plants. We have developed a new seed-based assay for meiotic recombination in Arabidopsis. The assay is based on the transformation of green and red fluorescent markers expressed

  4. Mek1/Mre4 is a master regulator of meiotic recombination in budding yeast

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    Nancy M. Hollingsworth

    2016-02-01

    Full Text Available Sexually reproducing organisms create gametes with half the somatic cell chromosome number so that fusion of gametes at fertilization does not change the ploidy of the cell. This reduction in chromosome number occurs by the specialized cell division of meiosis in which two rounds of chromosome segregation follow a single round of chromosome duplication. Meiotic crossovers formed between the non-sister chromatids of homologous chromosomes, combined with sister chromatid cohesion, physically connect homologs, thereby allowing proper segregation at the first meiotic division. Meiotic recombination is initiated by programmed double strand breaks (DSBs whose repair is highly regulated such that (1 there is a bias for recombination with homologs rather than sister chromatids, (2 crossovers are distributed throughout the genome by a process called interference, (3 crossover homeostasis regulates the balance between crossover and non-crossover repair to maintain a critical number of crossovers and (4 each pair of homologs receives at least one crossover. It was previously known that the imposition of interhomolog bias in budding yeast requires meiosis-specific modifications to the DNA damage response and the local activation of the meiosis-specific Mek1/Mre4 (hereafter Mek1 kinase at DSBs. However, because inactivation of Mek1 results in intersister, rather than interhomolog DSB repair, whether Mek1 had a role in interhomolog pathway choice was unknown. A recent study by Chen et al. (2015 reveals that Mek1 indirectly regulates the crossover/non-crossover decision between homologs as well as genetic interference. It does this by enabling phosphorylation of Zip1, the meiosis-specific transverse filament protein of the synaptonemal complex (SC, by the conserved cell cycle kinase, Cdc7-Dbf4 (DDK. These results suggest that Mek1 is a “master regulator” of meiotic recombination in budding yeast.

  5. VDE-initiated intein homing in Saccharomyces cerevisiae proceeds in a meiotic recombination-like manner.

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    Fukuda, Tomoyuki; Nogami, Satoru; Ohya, Yoshikazu

    2003-07-01

    Inteins and group I introns found in prokaryotic and eukaryotic organisms occasionally behave as mobile genetic elements. During meiosis of the yeast Saccharomyces cerevisiae, the site-specific endonuclease encoded by VMA1 intein, VDE, triggers a single double-strand break (DSB) at an inteinless allele, leading to VMA1 intein homing. Besides the accumulating information on the in vitro activity of VDE, very little has been known about the molecular mechanism of intein homing in yeast nucleus. We developed an assay to detect the product of VMA1 intein homing in yeast genome. We analysed mutant phenotypes of RecA homologs, Rad51p and Dmc1p, and their interacting proteins, Rad54p and Tid1p, and found that they all play critical roles in intein inheritance. The absence of DSB end processing proteins, Sae2p and those in the Mre11-Rad50-Xrs2 complex, also causes partial reduction in homing efficiency. As with meiotic recombination, crossover events are frequently observed during intein homing. We also observed that the absence of premeiotic DNA replication caused by hydroxyurea (HU) or clb5delta clb6delta mutation reduces VDE-mediated DSBs. The repairing system working in intein homing shares molecular machinery with meiotic recombination induced by Spo11p. Moreover, like Spo11p-induced DNA cleavage, premeiotic DNA replication is a prerequisite for a VDE-induced DSB. VMA1 intein thus utilizes several host factors involved in meiotic and recombinational processes to spread its genetic information and guarantee its progeny through establishment of a parasitic relationship with the organism.

  6. The temporal response of recombination events to gamma radiation of meiotic cells in Sordaria brevicollis.

    Science.gov (United States)

    Lewis, L A

    1982-01-01

    The temporal frequencies of different stages of prophase I were determined cytologically in Sordaria brevicollis (Olive and Fantini) as the basis for ascertaining the degree of synchrony in meiosis in this ascomycete. Croziers, karyogamy-zygotene and pachytene asci were shown to be in significant majorities at three distinct periods of the meiotic cycle. The response of recombination frequency to ionizing radiation was examined for the entire meiotic cycle. Three radiosensitive periods were determined. This response, which correlated temporally with each of the three peaks in ascal frequency, is interpreted as showing that the meiotic cycle of this organism is divided into periods of recombination commitment (radiation reduced frequencies) during the pre-meiotic S phase and recombination consummation (radiation induced frequencies) during zygotene and pachytene. The results are discussed in the context of the time at which recombination is consummated in eukaryotes such as yeast and Drosophila.

  7. Contrasted patterns of crossover and non-crossover at Arabidopsis thaliana meiotic recombination hotspots.

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    Jan Drouaud

    2013-11-01

    Full Text Available The vast majority of meiotic recombination events (crossovers (COs and non-crossovers (NCOs cluster in narrow hotspots surrounded by large regions devoid of recombinational activity. Here, using a new molecular approach in plants, called "pollen-typing", we detected and characterized hundreds of CO and NCO molecules in two different hotspot regions in Arabidopsis thaliana. This analysis revealed that COs are concentrated in regions of a few kilobases where their rates reach up to 50 times the genome average. The hotspots themselves tend to cluster in regions less than 8 kilobases in size with overlapping CO distribution. Non-crossover (NCO events also occurred in the two hotspots but at very different levels (local CO/NCO ratios of 1/1 and 30/1 and their track lengths were quite small (a few hundred base pairs. We also showed that the ZMM protein MSH4 plays a role in CO formation and somewhat unexpectedly we also found that it is involved in the generation of NCOs but with a different level of effect. Finally, factors acting in cis and in trans appear to shape the rate and distribution of COs at meiotic recombination hotspots.

  8. Sordaria, a model system to uncover links between meiotic pairing and recombination.

    Science.gov (United States)

    Zickler, Denise; Espagne, Eric

    2016-06-01

    The mycelial fungus Sordaria macrospora was first used as experimental system for meiotic recombination. This review shows that it provides also a powerful cytological system for dissecting chromosome dynamics in wild-type and mutant meioses. Fundamental cytogenetic findings include: (1) the identification of presynaptic alignment as a key step in pairing of homologous chromosomes. (2) The discovery that biochemical complexes that mediate recombination at the DNA level concomitantly mediate pairing of homologs. (3) This pairing process involves not only resolution but also avoidance of chromosomal entanglements and the resolution system includes dissolution of constraining DNA recombination interactions, achieved by a unique role of Mlh1. (4) Discovery that the central components of the synaptonemal complex directly mediate the re-localization of the recombination proteins from on-axis to in-between homologue axis positions. (5) Identification of putative STUbL protein Hei10 as a structure-based signal transduction molecule that coordinates progression and differentiation of recombinational interactions at multiple stages. (6) Discovery that a single interference process mediates both nucleation of the SC and designation of crossover sites, thereby ensuring even spacing of both features. (7) Discovery of local modulation of sister-chromatid cohesion at sites of crossover recombination. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Scrambling Eggs: Meiotic Drive and the Evolution of Female Recombination Rates

    Science.gov (United States)

    Brandvain, Yaniv; Coop, Graham

    2012-01-01

    Theories to explain the prevalence of sex and recombination have long been a central theme of evolutionary biology. Yet despite decades of attention dedicated to the evolution of sex and recombination, the widespread pattern of sex differences in the recombination rate is not well understood and has received relatively little theoretical attention. Here, we argue that female meiotic drivers—alleles that increase in frequency by exploiting the asymmetric cell division of oogenesis—present a potent selective pressure favoring the modification of the female recombination rate. Because recombination plays a central role in shaping patterns of variation within and among dyads, modifiers of the female recombination rate can function as potent suppressors or enhancers of female meiotic drive. We show that when female recombination modifiers are unlinked to female drivers, recombination modifiers that suppress harmful female drive can spread. By contrast, a recombination modifier tightly linked to a driver can increase in frequency by enhancing female drive. Our results predict that rapidly evolving female recombination rates, particularly around centromeres, should be a common outcome of meiotic drive. We discuss how selection to modify the efficacy of meiotic drive may contribute to commonly observed patterns of sex differences in recombination. PMID:22143919

  10. LDsplit: screening for cis-regulatory motifs stimulating meiotic recombination hotspots by analysis of DNA sequence polymorphisms.

    Science.gov (United States)

    Yang, Peng; Wu, Min; Guo, Jing; Kwoh, Chee Keong; Przytycka, Teresa M; Zheng, Jie

    2014-02-17

    As a fundamental genomic element, meiotic recombination hotspot plays important roles in life sciences. Thus uncovering its regulatory mechanisms has broad impact on biomedical research. Despite the recent identification of the zinc finger protein PRDM9 and its 13-mer binding motif as major regulators for meiotic recombination hotspots, other regulators remain to be discovered. Existing methods for finding DNA sequence motifs of recombination hotspots often rely on the enrichment of co-localizations between hotspots and short DNA patterns, which ignore the cross-individual variation of recombination rates and sequence polymorphisms in the population. Our objective in this paper is to capture signals encoded in genetic variations for the discovery of recombination-associated DNA motifs. Recently, an algorithm called "LDsplit" has been designed to detect the association between single nucleotide polymorphisms (SNPs) and proximal meiotic recombination hotspots. The association is measured by the difference of population recombination rates at a hotspot between two alleles of a candidate SNP. Here we present an open source software tool of LDsplit, with integrative data visualization for recombination hotspots and their proximal SNPs. Applying LDsplit on SNPs inside an established 7-mer motif bound by PRDM9 we observed that SNP alleles preserving the original motif tend to have higher recombination rates than the opposite alleles that disrupt the motif. Running on SNP windows around hotspots each containing an occurrence of the 7-mer motif, LDsplit is able to guide the established motif finding algorithm of MEME to recover the 7-mer motif. In contrast, without LDsplit the 7-mer motif could not be identified. LDsplit is a software tool for the discovery of cis-regulatory DNA sequence motifs stimulating meiotic recombination hotspots by screening and narrowing down to hotspot associated SNPs. It is the first computational method that utilizes the genetic variation of

  11. Correlation between pairing initiation sites, recombination nodules and meiotic recombination in Sordaria macrospora.

    Science.gov (United States)

    Zickler, D; Moreau, P J; Huynh, A D; Slezec, A M

    1992-09-01

    The decrease of meiotic exchanges (crossing over and conversion) in two mutants of Sordaria macrospora correlated strongly with a reduction of chiasmata and of both types of "recombination nodules." Serial section reconstruction electron microscopy was used to compare the synapsis pattern of meiotic prophase I in wild type and mutants. First, synapsis occurred but the number of synaptonemal complex initiation sites was reduced in both mutants. Second, this reduction was accompanied by, or resulted in, modifications of the pattern of synapsis. Genetic and synaptonemal complex maps were compared in three regions along one chromosome arm divided into well marked intervals. Reciprocal exchange frequencies and number of recombination nodules correlated in wild type in the three analyzed intervals, but disparity was found between the location of recombination nodules and exchanges in the mutants. Despite the twofold exchange decrease, sections of the genome such as the short arm of chromosome 2 and telomere regions were sheltered from nodule decrease and from pairing modifications. This indicated a certain amount of diversity in the control of these features and suggested that exchange frequency was dependent not only on the amount of effective pairing but also on the localization of the pairing sites, as revealed by the synaptonemal complex progression in the mutants.

  12. Conditional genomic rearrangement by designed meiotic recombination using VDE (PI-SceI) in yeast.

    Science.gov (United States)

    Fukuda, Tomoyuki; Ohya, Yoshikazu; Ohta, Kunihiro

    2007-10-01

    Meiotic recombination plays critical roles in the acquisition of genetic diversity and has been utilized for conventional breeding of livestock and crops. The frequency of meiotic recombination is normally low, and is extremely low in regions called "recombination cold domains". Here, we describe a new and highly efficient method to modulate yeast meiotic gene rearrangements using VDE (PI-SceI), an intein-encoded endonuclease that causes an efficient unidirectional meiotic gene conversion at its recognition sequence (VRS). We designed universal targeting vectors, by use of which the strain that inserts the VRS at a desired site is acquired. Meiotic induction of the strains provided unidirectional gene conversions and frequent genetic rearrangements of flanking genes with little impact on cell viability. This system thus opens the way for the designed modulation of meiotic gene rearrangements, regardless of recombinational activity of chromosomal domains. Finally, the VDE-VRS system enabled us to conduct meiosis-specific conditional knockout of genes where VDE-initiated gene conversion disrupts the target gene during meiosis, serving as a novel approach to examine the functions of genes during germination of resultant spores.

  13. mlh3 mutations in baker's yeast alter meiotic recombination outcomes by increasing noncrossover events genome-wide.

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    Najla Al-Sweel

    2017-08-01

    Full Text Available Mlh1-Mlh3 is an endonuclease hypothesized to act in meiosis to resolve double Holliday junctions into crossovers. It also plays a minor role in eukaryotic DNA mismatch repair (MMR. To understand how Mlh1-Mlh3 functions in both meiosis and MMR, we analyzed in baker's yeast 60 new mlh3 alleles. Five alleles specifically disrupted MMR, whereas one (mlh3-32 specifically disrupted meiotic crossing over. Mlh1-mlh3 representatives for each class were purified and characterized. Both Mlh1-mlh3-32 (MMR+, crossover- and Mlh1-mlh3-45 (MMR-, crossover+ displayed wild-type endonuclease activities in vitro. Msh2-Msh3, an MSH complex that acts with Mlh1-Mlh3 in MMR, stimulated the endonuclease activity of Mlh1-mlh3-32 but not Mlh1-mlh3-45, suggesting that Mlh1-mlh3-45 is defective in MSH interactions. Whole genome recombination maps were constructed for wild-type and MMR+ crossover-, MMR- crossover+, endonuclease defective and null mlh3 mutants in an S288c/YJM789 hybrid background. Compared to wild-type, all of the mlh3 mutants showed increases in the number of noncrossover events, consistent with recombination intermediates being resolved through alternative recombination pathways. Our observations provide a structure-function map for Mlh3 that reveals the importance of protein-protein interactions in regulating Mlh1-Mlh3's enzymatic activity. They also illustrate how defective meiotic components can alter the fate of meiotic recombination intermediates, providing new insights for how meiotic recombination pathways are regulated.

  14. Meiotic recombination breakpoints are associated with open chromatin and enriched with Stowaway transposons in potato

    Science.gov (United States)

    Meiotic recombination is the foundation for genetic variation in natural and artificial populations of eukaryotes. Although genetic recombination maps have been developed in numerous plant species since late the 1980s, very few of these maps have provided the necessary resolution needed to investiga...

  15. Meiotic recombination analyses of individual chromosomes in male domestic pigs (Sus scrofa domestica.

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    Nicolas Mary

    Full Text Available For the first time in the domestic pig, meiotic recombination along the 18 porcine autosomes was directly studied by immunolocalization of MLH1 protein. In total, 7,848 synaptonemal complexes from 436 spermatocytes were analyzed, and 13,969 recombination sites were mapped. Individual chromosomes for 113 of the 436 cells (representing 2,034 synaptonemal complexes were identified by immunostaining and fluorescence in situ hybridization (FISH. The average total length of autosomal synaptonemal complexes per cell was 190.3 µm, with 32.0 recombination sites (crossovers, on average, per cell. The number of crossovers and the lengths of the autosomal synaptonemal complexes showed significant intra- (i.e. between cells and inter-individual variations. The distributions of recombination sites within each chromosomal category were similar: crossovers in metacentric and submetacentric chromosomes were concentrated in the telomeric regions of the p- and q-arms, whereas two hotspots were located near the centromere and in the telomeric region of acrocentrics. Lack of MLH1 foci was mainly observed in the smaller chromosomes, particularly chromosome 18 (SSC18 and the sex chromosomes. All autosomes displayed positive interference, with a large variability between the chromosomes.

  16. Utilization during mitotic cell division of loci controlling meiotic recombination and disjunction in Drosophila melanogaster

    International Nuclear Information System (INIS)

    Baker, B.S.; Carpenter, A.T.C.; Ripoll, P.

    1978-01-01

    To inquire whether the loci identified by recombination-defective and disjunction-defective meiotic mutants in Drosophila are also utilized during mitotic cell division, the effects of 18 meiotic mutants (representing 13 loci) on mitotic chromosome stability have been examined genetically. To do this, meiotic-mutant-bearing flies heterozygous for recessive somatic cell markers were examined for the frequencies and types of spontaneous clones expressing the cell markers. In such flies, marked clones can arise via mitotic recombination, mutation, chromosome breakage, nondisjunction or chromosome loss, and clones from these different origins can be distinguished. In addition, meiotic mutants at nine loci have been examined for their effects on sensitivity to killing by uv and x rays. Mutants at six of the seven recombination-defective loci examined (mei-9, mei-41, c(3)G, mei-W68, mei-S282, mei-352, mei-218) cause mitotic chromosome instability in both sexes, whereas mutants at one locus (mei-218) do not affect mitotic chromosome stability. Thus many of the loci utilized during meiotic recombination also function in the chromosomal economy of mitotic cells

  17. BRIT1/MCPH1 is essential for mitotic and meiotic recombination DNA repair and maintaining genomic stability in mice.

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    Yulong Liang

    2010-01-01

    Full Text Available BRIT1 protein (also known as MCPH1 contains 3 BRCT domains which are conserved in BRCA1, BRCA2, and other important molecules involved in DNA damage signaling, DNA repair, and tumor suppression. BRIT1 mutations or aberrant expression are found in primary microcephaly patients as well as in cancer patients. Recent in vitro studies suggest that BRIT1/MCPH1 functions as a novel key regulator in the DNA damage response pathways. To investigate its physiological role and dissect the underlying mechanisms, we generated BRIT1(-/- mice and identified its essential roles in mitotic and meiotic recombination DNA repair and in maintaining genomic stability. Both BRIT1(-/- mice and mouse embryonic fibroblasts (MEFs were hypersensitive to gamma-irradiation. BRIT1(-/- MEFs and T lymphocytes exhibited severe chromatid breaks and reduced RAD51 foci formation after irradiation. Notably, BRIT1(-/- mice were infertile and meiotic homologous recombination was impaired. BRIT1-deficient spermatocytes exhibited a failure of chromosomal synapsis, and meiosis was arrested at late zygotene of prophase I accompanied by apoptosis. In mutant spermatocytes, DNA double-strand breaks (DSBs were formed, but localization of RAD51 or BRCA2 to meiotic chromosomes was severely impaired. In addition, we found that BRIT1 could bind to RAD51/BRCA2 complexes and that, in the absence of BRIT1, recruitment of RAD51 and BRCA2 to chromatin was reduced while their protein levels were not altered, indicating that BRIT1 is involved in mediating recruitment of RAD51/BRCA2 to the damage site. Collectively, our BRIT1-null mouse model demonstrates that BRIT1 is essential for maintaining genomic stability in vivo to protect the hosts from both programmed and irradiation-induced DNA damages, and its depletion causes a failure in both mitotic and meiotic recombination DNA repair via impairing RAD51/BRCA2's function and as a result leads to infertility and genomic instability in mice.

  18. Correlation of Meiotic DSB Formation and Transcription Initiation Around Fission Yeast Recombination Hotspots.

    Science.gov (United States)

    Yamada, Shintaro; Okamura, Mika; Oda, Arisa; Murakami, Hiroshi; Ohta, Kunihiro; Yamada, Takatomi

    2017-06-01

    Meiotic homologous recombination, a critical event for ensuring faithful chromosome segregation and creating genetic diversity, is initiated by programmed DNA double-strand breaks (DSBs) formed at recombination hotspots. Meiotic DSB formation is likely to be influenced by other DNA-templated processes including transcription, but how DSB formation and transcription interact with each other has not been understood well. In this study, we used fission yeast to investigate a possible interplay of these two events. A group of hotspots in fission yeast are associated with sequences similar to the cyclic AMP response element and activated by the ATF/CREB family transcription factor dimer Atf1-Pcr1. We first focused on one of those hotspots, ade6-3049 , and Atf1. Our results showed that multiple transcripts, shorter than the ade6 full-length messenger RNA, emanate from a region surrounding the ade6-3049 hotspot. Interestingly, we found that the previously known recombination-activation region of Atf1 is also a transactivation domain, whose deletion affected DSB formation and short transcript production at ade6-3049 These results point to a possibility that the two events may be related to each other at ade6-3049 In fact, comparison of published maps of meiotic transcripts and hotspots suggested that hotspots are very often located close to meiotically transcribed regions. These observations therefore propose that meiotic DSB formation in fission yeast may be connected to transcription of surrounding regions. Copyright © 2017 by the Genetics Society of America.

  19. Unisexual reproduction drives meiotic recombination and phenotypic and karyotypic plasticity in Cryptococcus neoformans.

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    Sheng Sun

    2014-12-01

    Full Text Available In fungi, unisexual reproduction, where sexual development is initiated without the presence of two compatible mating type alleles, has been observed in several species that can also undergo traditional bisexual reproduction, including the important human fungal pathogens Cryptococcus neoformans and Candida albicans. While unisexual reproduction has been well characterized qualitatively, detailed quantifications are still lacking for aspects of this process, such as the frequency of recombination during unisexual reproduction, and how this compares with bisexual reproduction. Here, we analyzed meiotic recombination during α-α unisexual and a-α bisexual reproduction of C. neoformans. We found that meiotic recombination operates in a similar fashion during both modes of sexual reproduction. Specifically, we observed that in α-α unisexual reproduction, the numbers of crossovers along the chromosomes during meiosis, recombination frequencies at specific chromosomal regions, as well as meiotic recombination hot and cold spots, are all similar to those observed during a-α bisexual reproduction. The similarity in meiosis is also reflected by the fact that phenotypic segregation among progeny collected from the two modes of sexual reproduction is also similar, with transgressive segregation being observed in both. Additionally, we found diploid meiotic progeny were also produced at similar frequencies in the two modes of sexual reproduction, and transient chromosomal loss and duplication likely occurs frequently and results in aneuploidy and loss of heterozygosity that can span entire chromosomes. Furthermore, in both α-α unisexual and a-α bisexual reproduction, we observed biased allele inheritance in regions on chromosome 4, suggesting the presence of fragile chromosomal regions that might be vulnerable to mitotic recombination. Interestingly, we also observed a crossover event that occurred within the MAT locus during α-α unisexual

  20. Unisexual Reproduction Drives Meiotic Recombination and Phenotypic and Karyotypic Plasticity in Cryptococcus neoformans

    Science.gov (United States)

    Sun, Sheng; Billmyre, R. Blake; Mieczkowski, Piotr A.; Heitman, Joseph

    2014-01-01

    In fungi, unisexual reproduction, where sexual development is initiated without the presence of two compatible mating type alleles, has been observed in several species that can also undergo traditional bisexual reproduction, including the important human fungal pathogens Cryptococcus neoformans and Candida albicans. While unisexual reproduction has been well characterized qualitatively, detailed quantifications are still lacking for aspects of this process, such as the frequency of recombination during unisexual reproduction, and how this compares with bisexual reproduction. Here, we analyzed meiotic recombination during α-α unisexual and a-α bisexual reproduction of C. neoformans. We found that meiotic recombination operates in a similar fashion during both modes of sexual reproduction. Specifically, we observed that in α-α unisexual reproduction, the numbers of crossovers along the chromosomes during meiosis, recombination frequencies at specific chromosomal regions, as well as meiotic recombination hot and cold spots, are all similar to those observed during a-α bisexual reproduction. The similarity in meiosis is also reflected by the fact that phenotypic segregation among progeny collected from the two modes of sexual reproduction is also similar, with transgressive segregation being observed in both. Additionally, we found diploid meiotic progeny were also produced at similar frequencies in the two modes of sexual reproduction, and transient chromosomal loss and duplication likely occurs frequently and results in aneuploidy and loss of heterozygosity that can span entire chromosomes. Furthermore, in both α-α unisexual and a-α bisexual reproduction, we observed biased allele inheritance in regions on chromosome 4, suggesting the presence of fragile chromosomal regions that might be vulnerable to mitotic recombination. Interestingly, we also observed a crossover event that occurred within the MAT locus during α-α unisexual reproduction. Our results

  1. Meiotic homoeologous recombination-based alien gene introgression in the genomics era of wheat

    Science.gov (United States)

    Wheat (Triticum spp.) has a narrow genetic basis due to its allopolyploid origin. However, wheat has numerous wild relatives usable for expanding genetic variability of its genome through meiotic homoeologous recombination. Traditionally, laborious cytological analyses have been employed to detect h...

  2. PRDM9 drives evolutionary erosion of hotspots in Mus musculus through haplotype-specific initiation of meiotic recombination.

    Science.gov (United States)

    Baker, Christopher L; Kajita, Shimpei; Walker, Michael; Saxl, Ruth L; Raghupathy, Narayanan; Choi, Kwangbom; Petkov, Petko M; Paigen, Kenneth

    2015-01-01

    Meiotic recombination generates new genetic variation and assures the proper segregation of chromosomes in gametes. PRDM9, a zinc finger protein with histone methyltransferase activity, initiates meiotic recombination by binding DNA at recombination hotspots and directing the position of DNA double-strand breaks (DSB). The DSB repair mechanism suggests that hotspots should eventually self-destruct, yet genome-wide recombination levels remain constant, a conundrum known as the hotspot paradox. To test if PRDM9 drives this evolutionary erosion, we measured activity of the Prdm9Cst allele in two Mus musculus subspecies, M.m. castaneus, in which Prdm9Cst arose, and M.m. domesticus, into which Prdm9Cst was introduced experimentally. Comparing these two strains, we find that haplotype differences at hotspots lead to qualitative and quantitative changes in PRDM9 binding and activity. Using Mus spretus as an outlier, we found most variants affecting PRDM9Cst binding arose and were fixed in M.m. castaneus, suppressing hotspot activity. Furthermore, M.m. castaneus×M.m. domesticus F1 hybrids exhibit novel hotspots, with large haplotype biases in both PRDM9 binding and chromatin modification. These novel hotspots represent sites of historic evolutionary erosion that become activated in hybrids due to crosstalk between one parent's Prdm9 allele and the opposite parent's chromosome. Together these data support a model where haplotype-specific PRDM9 binding directs biased gene conversion at hotspots, ultimately leading to hotspot erosion.

  3. Association of poly-purine/poly-pyrimidine sequences with meiotic recombination hot spots

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    Pitt Joel PW

    2006-07-01

    Full Text Available Abstract Background Meiotic recombination events have been found to concentrate in 1–2.5 kilo base regions, but these recombination hot spots do not share a consensus sequence and why they occur at specific sites is not fully understood. Some previous evidence suggests that poly-purine/poly-pyrimidine (poly-pu/py tracts (PPTs, a class of sequence with distinctive biochemical properties, could be involved in recombination, but no general association of PPTs with meiotic recombination hot spots has previously been reported. Results We used computational methods to investigate in detail the relationship between PPTs and hot spots. We show statistical associations of PPT frequency with hot spots of meiotic recombination initiating lesions, double-strand breaks, in the genome of the yeast S. cerevisiae and with experimentally well characterized human meiotic recombination hot spots. Supporting a possible role of poly-pu/py-rich sequences in hot spot recombination, we also found that all three single nucleotide polymorphisms previously shown to be associated with human hot spot activity changes occur within sequence contexts of 14 bp or longer that are 85% or more poly-pu/py and at least 70% G/C. These polymorphisms are all close to the hot spot mid points. Comparing the sequences of experimentally characterized human hot spots with the orthologous regions of the chimpanzee genome previously shown not to contain hot spots, we found that in all five cases in which comparisons for the hot spot central regions are possible with publicly available sequence data, there are differences near the human hot spot mid points within sequences 14 bp or longer consisting of more than 80% poly-pu/py and at least 50% G/C. Conclusion Our results, along with previous evidence for the unique biochemical properties and recombination-stimulating potential of poly-pu/py-rich sequences, suggest that the possible functional involvement of this type of sequence in meiotic

  4. Functional Roles of Acetylated Histone Marks at Mouse Meiotic Recombination Hot Spots

    Science.gov (United States)

    Wu, Zhen; Fallahi, Mohammad; Ouizem, Souad; Liu, Qin; Li, Weimin; Costi, Roberta; Roush, William R.; Bois, Philippe R. J.

    2016-01-01

    ABSTRACT Meiotic recombination initiates following the formation of DNA double-strand breaks (DSBs) by the Spo11 endonuclease early in prophase I, at discrete regions in the genome coined “hot spots.” In mammals, meiotic DSB site selection is directed in part by sequence-specific binding of PRDM9, a polymorphic histone H3 (H3K4Me3) methyltransferase. However, other chromatin features needed for meiotic hot spot specification are largely unknown. Here we show that the recombinogenic cores of active hot spots in mice harbor several histone H3 and H4 acetylation and methylation marks that are typical of open, active chromatin. Further, deposition of these open chromatin-associated histone marks is dynamic and is manifest at spermatogonia and/or pre-leptotene-stage cells, which facilitates PRDM9 binding and access for Spo11 to direct the formation of DSBs, which are initiated at the leptotene stage. Importantly, manipulating histone acetylase and deacetylase activities established that histone acetylation marks are necessary for both hot spot activity and crossover resolution. We conclude that there are functional roles for histone acetylation marks at mammalian meiotic recombination hot spots. PMID:27821479

  5. High-Resolution Patterns of Meiotic Recombination across the Human Major Histocompatibility Complex

    Science.gov (United States)

    Cullen, Michael; Perfetto, Stephen P.; Klitz, William; Nelson, George; Carrington, Mary

    2002-01-01

    Definitive characteristics of meiotic recombination events over large (i.e., >1 Mb) segments of the human genome remain obscure, yet they are essential for establishing the haplotypic structure of the genome and for efficient mapping of complex traits. We present a high-resolution map of recombination at the kilobase level across a 3.3-Mb interval encompassing the major histocompatibility complex (MHC). Genotyping of 20,031 single sperm from 12 individuals resulted in the identification and fine mapping of 325 recombinant chromosomes within genomic intervals as small as 7 kb. Several principal characteristics of recombination in this region were observed: (1) rates of recombination can differ significantly between individuals; (2) intense hot spots of recombination occur at least every 0.8 Mb but are not necessarily evenly spaced; (3) distribution in the location of recombination events can differ significantly among individuals; (4) between hot spots, low levels of recombination occur fairly evenly across 100-kb segments, suggesting the presence of warm spots of recombination; and (5) specific sequence motifs associate significantly with recombination distribution. These data provide a plausible model for recombination patterns of the human genome overall. PMID:12297984

  6. Meiotic sister chromatid cohesion and recombination in two filamentous fungi

    NARCIS (Netherlands)

    Heemst, van D.

    2000-01-01

    Homologous recombination and sister chromatid cohesion play important roles in the maintenance of genome integrity and the fidelity of chromosome segregation in mitosis and meiosis. Within the living cell, the integrity of the DNA is threatened by various factors that cause DNA-lesions, of

  7. CENTRAL REGION COMPONENT1, a Novel Synaptonemal Complex Component, Is Essential for Meiotic Recombination Initiation in Rice[C][W

    Science.gov (United States)

    Miao, Chunbo; Tang, Ding; Zhang, Honggen; Wang, Mo; Li, Yafei; Tang, Shuzhu; Yu, Hengxiu; Gu, Minghong; Cheng, Zhukuan

    2013-01-01

    In meiosis, homologous recombination entails programmed DNA double-strand break (DSB) formation and synaptonemal complex (SC) assembly coupled with the DSB repair. Although SCs display extensive structural conservation among species, their components identified are poorly conserved at the sequence level. Here, we identified a novel SC component, designated CENTRAL REGION COMPONENT1 (CRC1), in rice (Oryza sativa). CRC1 colocalizes with ZEP1, the rice SC transverse filament protein, to the central region of SCs in a mutually dependent fashion. Consistent with this colocalization, CRC1 interacts with ZEP1 in yeast two-hybrid assays. CRC1 is orthologous to Saccharomyces cerevisiae pachytene checkpoint2 (Pch2) and Mus musculus THYROID RECEPTOR-INTERACTING PROTEIN13 (TRIP13) and may be a conserved SC component. Additionally, we provide evidence that CRC1 is essential for meiotic DSB formation. CRC1 interacts with HOMOLOGOUS PAIRING ABERRATION IN RICE MEIOSIS1 (PAIR1) in vitro, suggesting that these proteins act as a complex to promote DSB formation. PAIR2, the rice ortholog of budding yeast homolog pairing1, is required for homologous chromosome pairing. We found that CRC1 is also essential for the recruitment of PAIR2 onto meiotic chromosomes. The roles of CRC1 identified here have not been reported for Pch2 or TRIP13. PMID:23943860

  8. Central region component1, a novel synaptonemal complex component, is essential for meiotic recombination initiation in rice.

    Science.gov (United States)

    Miao, Chunbo; Tang, Ding; Zhang, Honggen; Wang, Mo; Li, Yafei; Tang, Shuzhu; Yu, Hengxiu; Gu, Minghong; Cheng, Zhukuan

    2013-08-01

    In meiosis, homologous recombination entails programmed DNA double-strand break (DSB) formation and synaptonemal complex (SC) assembly coupled with the DSB repair. Although SCs display extensive structural conservation among species, their components identified are poorly conserved at the sequence level. Here, we identified a novel SC component, designated central region component1 (CRC1), in rice (Oryza sativa). CRC1 colocalizes with ZEP1, the rice SC transverse filament protein, to the central region of SCs in a mutually dependent fashion. Consistent with this colocalization, CRC1 interacts with ZEP1 in yeast two-hybrid assays. CRC1 is orthologous to Saccharomyces cerevisiae pachytene checkpoint2 (Pch2) and Mus musculus THYROID receptor-interacting protein13 (TRIP13) and may be a conserved SC component. Additionally, we provide evidence that CRC1 is essential for meiotic DSB formation. CRC1 interacts with homologous pairing aberration in rice meiosis1 (PAIR1) in vitro, suggesting that these proteins act as a complex to promote DSB formation. PAIR2, the rice ortholog of budding yeast homolog pairing1, is required for homologous chromosome pairing. We found that CRC1 is also essential for the recruitment of PAIR2 onto meiotic chromosomes. The roles of CRC1 identified here have not been reported for Pch2 or TRIP13.

  9. Identification of new genes required for meiotic recombination in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Ajimura, M.; Lee, S.H.; Ogawa, H.

    1993-01-01

    Mutants defective in meiotic recombination were isolated from a disomic haploid strain of Saccharomyces cerevisiae by examining recombination within the leu2 and his4 heteroalleles located on chromosome III. The mutants were classified into two new complementation groups (MRE2 and MRE11) and eight previously identified groups, which include SPO11, HOP1, REC114, MRE4/MEK1 and genes in the RAD52 epistasis group. All of the mutants, in which the mutations in the new complementation groups are homozygous and diploid, can undergo premeiotic DNA synthesis and produce spores. The spores are, however, not viable. The mre2 and mre11 mutants produce viable spores in a spo13 background, in which meiosis I is bypassed, suggesting that these mutants are blocked at an early step in meiotic recombination. The mre2 mutant does not exhibit any unusual phenotype during mitosis and it is, thus, considered to have a mutation in a meiosis-specific gene. By contrast, the mre11 mutant is sensitive to damage to DNA by methyl methanesulfonate and exhibits a hyperrecombination phenotype in mitosis. Among six alleles of HOP1 that were isolated, an unusual pattern of intragenic complementation was observed

  10. Meiotic Clade AAA ATPases: Protein Polymer Disassembly Machines.

    Science.gov (United States)

    Monroe, Nicole; Hill, Christopher P

    2016-05-08

    Meiotic clade AAA ATPases (ATPases associated with diverse cellular activities), which were initially grouped on the basis of phylogenetic classification of their AAA ATPase cassette, include four relatively well characterized family members, Vps4, spastin, katanin and fidgetin. These enzymes all function to disassemble specific polymeric protein structures, with Vps4 disassembling the ESCRT-III polymers that are central to the many membrane-remodeling activities of the ESCRT (endosomal sorting complexes required for transport) pathway and spastin, katanin p60 and fidgetin affecting multiple aspects of cellular dynamics by severing microtubules. They share a common domain architecture that features an N-terminal MIT (microtubule interacting and trafficking) domain followed by a single AAA ATPase cassette. Meiotic clade AAA ATPases function as hexamers that can cycle between the active assembly and inactive monomers/dimers in a regulated process, and they appear to disassemble their polymeric substrates by translocating subunits through the central pore of their hexameric ring. Recent studies with Vps4 have shown that nucleotide-induced asymmetry is a requirement for substrate binding to the pore loops and that recruitment to the protein lattice via MIT domains also relieves autoinhibition and primes the AAA ATPase cassettes for substrate binding. The most striking, unifying feature of meiotic clade AAA ATPases may be their MIT domain, which is a module that is found in a wide variety of proteins that localize to ESCRT-III polymers. Spastin also displays an adjacent microtubule binding sequence, and the presence of both ESCRT-III and microtubule binding elements may underlie the recent findings that the ESCRT-III disassembly function of Vps4 and the microtubule-severing function of spastin, as well as potentially katanin and fidgetin, are highly coordinated. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Controlling meiotic recombinational repair - specifying the roles of ZMMs, Sgs1 and Mus81/Mms4 in crossover formation.

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    Ashwini Oke

    2014-10-01

    Full Text Available Crossovers (COs play a critical role in ensuring proper alignment and segregation of homologous chromosomes during meiosis. How the cell balances recombination between CO vs. noncrossover (NCO outcomes is not completely understood. Further lacking is what constrains the extent of DNA repair such that multiple events do not arise from a single double-strand break (DSB. Here, by interpreting signatures that result from recombination genome-wide, we find that synaptonemal complex proteins promote crossing over in distinct ways. Our results suggest that Zip3 (RNF212 promotes biased cutting of the double Holliday-junction (dHJ intermediate whereas surprisingly Msh4 does not. Moreover, detailed examination of conversion tracts in sgs1 and mms4-md mutants reveal distinct aberrant recombination events involving multiple chromatid invasions. In sgs1 mutants, these multiple invasions are generally multichromatid involving 3-4 chromatids; in mms4-md mutants the multiple invasions preferentially resolve into one or two chromatids. Our analysis suggests that Mus81/Mms4 (Eme1, rather than just being a minor resolvase for COs is crucial for both COs and NCOs in preventing chromosome entanglements by removing 3'- flaps to promote second-end capture. Together our results force a reevaluation of how key recombination enzymes collaborate to specify the outcome of meiotic DNA repair.

  12. Sexual antagonism and meiotic drive cause stable linkage disequilibrium and favour reduced recombination on the X chromosome.

    Science.gov (United States)

    Rydzewski, W T; Carioscia, S A; Liévano, G; Lynch, V D; Patten, M M

    2016-06-01

    Sexual antagonism and meiotic drive are sex-specific evolutionary forces with the potential to shape genomic architecture. Previous theory has found that pairing two sexually antagonistic loci or combining sexual antagonism with meiotic drive at linked autosomal loci augments genetic variation, produces stable linkage disequilibrium (LD) and favours reduced recombination. However, the influence of these two forces has not been examined on the X chromosome, which is thought to be enriched for sexual antagonism and meiotic drive. We investigate the evolution of the X chromosome under both sexual antagonism and meiotic drive with two models: in one, both loci experience sexual antagonism; in the other, we pair a meiotic drive locus with a sexually antagonistic locus. We find that LD arises between the two loci in both models, even when the two loci freely recombine in females and that driving haplotypes will be enriched for male-beneficial alleles, further skewing sex ratios in these populations. We introduce a new measure of LD, Dz', which accounts for population allele frequencies and is appropriate for instances where these are sex specific. Both models demonstrate that natural selection favours modifiers that reduce the recombination rate. These results inform observed patterns of congealment found on driving X chromosomes and have implications for patterns of natural variation and the evolution of recombination rates on the X chromosome. © 2016 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2016 European Society For Evolutionary Biology.

  13. Asy2/Mer2: an evolutionarily conserved mediator of meiotic recombination, pairing, and global chromosome compaction.

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    Tessé, Sophie; Bourbon, Henri-Marc; Debuchy, Robert; Budin, Karine; Dubois, Emeline; Liangran, Zhang; Antoine, Romain; Piolot, Tristan; Kleckner, Nancy; Zickler, Denise; Espagne, Eric

    2017-09-15

    Meiosis is the cellular program by which a diploid cell gives rise to haploid gametes for sexual reproduction. Meiotic progression depends on tight physical and functional coupling of recombination steps at the DNA level with specific organizational features of meiotic-prophase chromosomes. The present study reveals that every step of this coupling is mediated by a single molecule: Asy2/Mer2. We show that Mer2, identified so far only in budding and fission yeasts, is in fact evolutionarily conserved from fungi (Mer2/Rec15/Asy2/Bad42) to plants (PRD3/PAIR1) and mammals (IHO1). In yeasts, Mer2 mediates assembly of recombination-initiation complexes and double-strand breaks (DSBs). This role is conserved in the fungus Sordaria However, functional analysis of 13 mer2 mutants and successive localization of Mer2 to axis, synaptonemal complex (SC), and chromatin revealed, in addition, three further important functions. First, after DSB formation, Mer2 is required for pairing by mediating homolog spatial juxtaposition, with implications for crossover (CO) patterning/interference. Second, Mer2 participates in the transfer/maintenance and release of recombination complexes to/from the SC central region. Third, after completion of recombination, potentially dependent on SUMOylation, Mer2 mediates global chromosome compaction and post-recombination chiasma development. Thus, beyond its role as a recombinosome-axis/SC linker molecule, Mer2 has important functions in relation to basic chromosome structure. © 2017 Tessé et al.; Published by Cold Spring Harbor Laboratory Press.

  14. DNA Sequence-Mediated, Evolutionarily Rapid Redistribution of Meiotic Recombination Hotspots

    Science.gov (United States)

    Wahls, Wayne P.; Davidson, Mari K.

    2011-01-01

    Hotspots regulate the position and frequency of Spo11 (Rec12)-initiated meiotic recombination, but paradoxically they are suicidal and are somehow resurrected elsewhere in the genome. After the DNA sequence-dependent activation of hotspots was discovered in fission yeast, nearly two decades elapsed before the key realizations that (A) DNA site-dependent regulation is broadly conserved and (B) individual eukaryotes have multiple different DNA sequence motifs that activate hotspots. From our perspective, such findings provide a conceptually straightforward solution to the hotspot paradox and can explain other, seemingly complex features of meiotic recombination. We describe how a small number of single-base-pair substitutions can generate hotspots de novo and dramatically alter their distribution in the genome. This model also shows how equilibrium rate kinetics could maintain the presence of hotspots over evolutionary timescales, without strong selective pressures invoked previously, and explains why hotspots localize preferentially to intergenic regions and introns. The model is robust enough to account for all hotspots of humans and chimpanzees repositioned since their divergence from the latest common ancestor. PMID:22084420

  15. Topoisomerase 3alpha and RMI1 suppress somatic crossovers and are essential for resolution of meiotic recombination intermediates in Arabidopsis thaliana.

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    Frank Hartung

    2008-12-01

    Full Text Available Topoisomerases are enzymes with crucial functions in DNA metabolism. They are ubiquitously present in prokaryotes and eukaryotes and modify the steady-state level of DNA supercoiling. Biochemical analyses indicate that Topoisomerase 3alpha (TOP3alpha functions together with a RecQ DNA helicase and a third partner, RMI1/BLAP75, in the resolution step of homologous recombination in a process called Holliday Junction dissolution in eukaryotes. Apart from that, little is known about the role of TOP3alpha in higher eukaryotes, as knockout mutants show early lethality or strong developmental defects. Using a hypomorphic insertion mutant of Arabidopsis thaliana (top3alpha-2, which is viable but completely sterile, we were able to define three different functions of the protein in mitosis and meiosis. The top3alpha-2 line exhibits fragmented chromosomes during mitosis and sensitivity to camptothecin, suggesting an important role in chromosome segregation partly overlapping with that of type IB topoisomerases. Furthermore, AtTOP3alpha, together with AtRECQ4A and AtRMI1, is involved in the suppression of crossover recombination in somatic cells as well as DNA repair in both mammals and A. thaliana. Surprisingly, AtTOP3alpha is also essential for meiosis. The phenotype of chromosome fragmentation, bridges, and telophase I arrest can be suppressed by AtSPO11 and AtRAD51 mutations, indicating that the protein is required for the resolution of recombination intermediates. As Atrmi1 mutants have a similar meiotic phenotype to Attop3alpha mutants, both proteins seem to be involved in a mechanism safeguarding the entangling of homologous chromosomes during meiosis. The requirement of AtTOP3alpha and AtRMI1 in a late step of meiotic recombination strongly hints at the possibility that the dissolution of double Holliday Junctions via a hemicatenane intermediate is indeed an indispensable step of meiotic recombination.

  16. Recombinant Collagenlike Proteins

    Science.gov (United States)

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

  17. LDSplitDB: a database for studies of meiotic recombination hotspots in MHC using human genomic data.

    Science.gov (United States)

    Guo, Jing; Chen, Hao; Yang, Peng; Lee, Yew Ti; Wu, Min; Przytycka, Teresa M; Kwoh, Chee Keong; Zheng, Jie

    2018-04-20

    Meiotic recombination happens during the process of meiosis when chromosomes inherited from two parents exchange genetic materials to generate chromosomes in the gamete cells. The recombination events tend to occur in narrow genomic regions called recombination hotspots. Its dysregulation could lead to serious human diseases such as birth defects. Although the regulatory mechanism of recombination events is still unclear, DNA sequence polymorphisms have been found to play crucial roles in the regulation of recombination hotspots. To facilitate the studies of the underlying mechanism, we developed a database named LDSplitDB which provides an integrative and interactive data mining and visualization platform for the genome-wide association studies of recombination hotspots. It contains the pre-computed association maps of the major histocompatibility complex (MHC) region in the 1000 Genomes Project and the HapMap Phase III datasets, and a genome-scale study of the European population from the HapMap Phase II dataset. Besides the recombination profiles, related data of genes, SNPs and different types of epigenetic modifications, which could be associated with meiotic recombination, are provided for comprehensive analysis. To meet the computational requirement of the rapidly increasing population genomics data, we prepared a lookup table of 400 haplotypes for recombination rate estimation using the well-known LDhat algorithm which includes all possible two-locus haplotype configurations. To the best of our knowledge, LDSplitDB is the first large-scale database for the association analysis of human recombination hotspots with DNA sequence polymorphisms. It provides valuable resources for the discovery of the mechanism of meiotic recombination hotspots. The information about MHC in this database could help understand the roles of recombination in human immune system. DATABASE URL: http://histone.scse.ntu.edu.sg/LDSplitDB.

  18. Direct and indirect control of the initiation of meiotic recombination by DNA damage checkpoint mechanisms in budding yeast.

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    Bilge Argunhan

    Full Text Available Meiotic recombination plays an essential role in the proper segregation of chromosomes at meiosis I in many sexually reproducing organisms. Meiotic recombination is initiated by the scheduled formation of genome-wide DNA double-strand breaks (DSBs. The timing of DSB formation is strictly controlled because unscheduled DSB formation is detrimental to genome integrity. Here, we investigated the role of DNA damage checkpoint mechanisms in the control of meiotic DSB formation using budding yeast. By using recombination defective mutants in which meiotic DSBs are not repaired, the effect of DNA damage checkpoint mutations on DSB formation was evaluated. The Tel1 (ATM pathway mainly responds to unresected DSB ends, thus the sae2 mutant background in which DSB ends remain intact was employed. On the other hand, the Mec1 (ATR pathway is primarily used when DSB ends are resected, thus the rad51 dmc1 double mutant background was employed in which highly resected DSBs accumulate. In order to separate the effect caused by unscheduled cell cycle progression, which is often associated with DNA damage checkpoint defects, we also employed the ndt80 mutation which permanently arrests the meiotic cell cycle at prophase I. In the absence of Tel1, DSB formation was reduced in larger chromosomes (IV, VII, II and XI whereas no significant reduction was found in smaller chromosomes (III and VI. On the other hand, the absence of Rad17 (a critical component of the ATR pathway lead to an increase in DSB formation (chromosomes VII and II were tested. We propose that, within prophase I, the Tel1 pathway facilitates DSB formation, especially in bigger chromosomes, while the Mec1 pathway negatively regulates DSB formation. We also identified prophase I exit, which is under the control of the DNA damage checkpoint machinery, to be a critical event associated with down-regulating meiotic DSB formation.

  19. Mapping genes by meiotic and UV-induced mitotic recombination in Coprinus cinereus

    International Nuclear Information System (INIS)

    Amirkhanian, J.D.; Cowan, J.W.

    1985-01-01

    Three morphological mutants in Coprinus cinereus—one spontaneous (den-2) and two chemically induced (zigand sta)—were assigned to linkage groups and utilized in meiotic and mitotic mapping. Mutants den-2 and zig belong to linkage group III, den-2 being close to the centromere and about 20 map units (mu) from zig. The mutant sta in linkage group ‘G’ is at a distance of about 37 mu from ade-3. Mitotic mapping confirmed the gene order in linkage group III and provided evidence that trp-2 in linkage group ‘G’ was between the centromere and ade-3. These morphological mutants are compact in colony growth and therefore suited to high-density plating. The rarity of spontaneously occurring mitotic segregants suggests that diploids of Coprinus cinereus, heterozygous for morphoiogical markers in repuision, could serve as useful test systems for rapid screening of chemical mutagen/carcinogens via mitotic recombination studies

  20. Acetylated Histone H3K9 is associated with meiotic recombination hotspots, and plays a role in recombination redundantly with other factors including the H3K4 methylase Set1 in fission yeast

    Science.gov (United States)

    Yamada, Shintaro; Ohta, Kunihiro; Yamada, Takatomi

    2013-01-01

    Histone modifications are associated with meiotic recombination hotspots, discrete sites with augmented recombination frequency. For example, trimethylation of histone H3 lysine4 (H3K4me3) marks most hotspots in budding yeast and mouse. Modified histones are known to regulate meiotic recombination partly by promoting DNA double-strand break (DSB) formation at hotspots, but the role and precise landscape of involved modifications remain unclear. Here, we studied hotspot-associated modifications in fission yeast and found general features: acetylation of H3 lysine9 (H3K9ac) is elevated, and H3K4me3 is not significantly enriched. Mutating H3K9 to non-acetylatable alanine mildly reduced levels of the DSB-inducing protein Rec12 (the fission yeast homologue of Spo11) and DSB at hotspots, indicating that H3K9ac may be involved in DSB formation by enhancing the interaction between Rec12 and hotspots. In addition, we found that the lack of the H3K4 methyltransferase Set1 generally increased Rec12 binding to chromatin but partially reduced DSB formation at some loci, suggesting that Set1 is also involved in DSB formation. These results suggest that meiotic DSB formation is redundantly regulated by multiple chromatin-related factors including H3K9ac and Set1 in fission yeast. PMID:23382177

  1. Variation in genome-wide levels of meiotic recombination is established at the onset of prophase in mammalian males.

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    Brian Baier

    2014-01-01

    Full Text Available Segregation of chromosomes during the first meiotic division relies on crossovers established during prophase. Although crossovers are strictly regulated so that at least one occurs per chromosome, individual variation in crossover levels is not uncommon. In an analysis of different inbred strains of male mice, we identified among-strain variation in the number of foci for the crossover-associated protein MLH1. We report studies of strains with "low" (CAST/EiJ, "medium" (C3H/HeJ, and "high" (C57BL/6J genome-wide MLH1 values to define factors responsible for this variation. We utilized immunofluorescence to analyze the number and distribution of proteins that function at different stages in the recombination pathway: RAD51 and DMC1, strand invasion proteins acting shortly after double-strand break (DSB formation, MSH4, part of the complex stabilizing double Holliday junctions, and the Bloom helicase BLM, thought to have anti-crossover activity. For each protein, we identified strain-specific differences that mirrored the results for MLH1; i.e., CAST/EiJ mice had the lowest values, C3H/HeJ mice intermediate values, and C57BL/6J mice the highest values. This indicates that differences in the numbers of DSBs (as identified by RAD51 and DMC1 are translated into differences in the number of crossovers, suggesting that variation in crossover levels is established by the time of DSB formation. However, DSBs per se are unlikely to be the primary determinant, since allelic variation for the DSB-inducing locus Spo11 resulted in differences in the numbers of DSBs but not the number of MLH1 foci. Instead, chromatin conformation appears to be a more important contributor, since analysis of synaptonemal complex length and DNA loop size also identified consistent strain-specific differences; i.e., crossover frequency increased with synaptonemal complex length and was inversely related to chromatin loop size. This indicates a relationship between recombination

  2. Local and sex-specific biases in crossover vs. noncrossover outcomes at meiotic recombination hot spots in mice

    Science.gov (United States)

    de Boer, Esther; Jasin, Maria; Keeney, Scott

    2015-01-01

    Meiotic recombination initiated by programmed double-strand breaks (DSBs) yields two types of interhomolog recombination products, crossovers and noncrossovers, but what determines whether a DSB will yield a crossover or noncrossover is not understood. In this study, we analyzed the influence of sex and chromosomal location on mammalian recombination outcomes by constructing fine-scale recombination maps in both males and females at two mouse hot spots located in different regions of the same chromosome. These include the most comprehensive maps of recombination hot spots in oocytes to date. One hot spot, located centrally on chromosome 1, behaved similarly in male and female meiosis: Crossovers and noncrossovers formed at comparable levels and ratios in both sexes. In contrast, at a distal hot spot, crossovers were recovered only in males even though noncrossovers were obtained at similar frequencies in both sexes. These findings reveal an example of extreme sex-specific bias in recombination outcome. We further found that estimates of relative DSB levels are surprisingly poor predictors of relative crossover frequencies between hot spots in males. Our results demonstrate that the outcome of mammalian meiotic recombination can be biased, that this bias can vary depending on location and cellular context, and that DSB frequency is not the only determinant of crossover frequency. PMID:26251527

  3. Disruption of CHTF18 causes defective meiotic recombination in male mice.

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    Karen M Berkowitz

    Full Text Available CHTF18 (chromosome transmission fidelity factor 18 is an evolutionarily conserved subunit of the Replication Factor C-like complex, CTF18-RLC. CHTF18 is necessary for the faithful passage of chromosomes from one daughter cell to the next during mitosis in yeast, and it is crucial for germline development in the fruitfly. Previously, we showed that mouse Chtf18 is expressed throughout the germline, suggesting a role for CHTF18 in mammalian gametogenesis. To determine the role of CHTF18 in mammalian germ cell development, we derived mice carrying null and conditional mutations in the Chtf18 gene. Chtf18-null males exhibit 5-fold decreased sperm concentrations compared to wild-type controls, resulting in subfertility. Loss of Chtf18 results in impaired spermatogenesis; spermatogenic cells display abnormal morphology, and the stereotypical arrangement of cells within seminiferous tubules is perturbed. Meiotic recombination is defective and homologous chromosomes separate prematurely during prophase I. Repair of DNA double-strand breaks is delayed and incomplete; both RAD51 and γH2AX persist in prophase I. In addition, MLH1 foci are decreased in pachynema. These findings demonstrate essential roles for CHTF18 in mammalian spermatogenesis and meiosis, and suggest that CHTF18 may function during the double-strand break repair pathway to promote the formation of crossovers.

  4. PRDM9 variation strongly influences recombination hot-spot activity and meiotic instability in humans.

    Science.gov (United States)

    Berg, Ingrid L; Neumann, Rita; Lam, Kwan-Wood G; Sarbajna, Shriparna; Odenthal-Hesse, Linda; May, Celia A; Jeffreys, Alec J

    2010-10-01

    PRDM9 has recently been identified as a likely trans regulator of meiotic recombination hot spots in humans and mice. PRDM9 contains a zinc finger array that, in humans, can recognize a short sequence motif associated with hot spots, with binding to this motif possibly triggering hot-spot activity via chromatin remodeling. We now report that human genetic variation at the PRDM9 locus has a strong effect on sperm hot-spot activity, even at hot spots lacking the sequence motif. Subtle changes within the zinc finger array can create hot-spot nonactivating or enhancing variants and can even trigger the appearance of a new hot spot, suggesting that PRDM9 is a major global regulator of hot spots in humans. Variation at the PRDM9 locus also influences aspects of genome instability-specifically, a megabase-scale rearrangement underlying two genomic disorders as well as minisatellite instability-implicating PRDM9 as a risk factor for some pathological genome rearrangements.

  5. Prdm9, a major determinant of meiotic recombination hotspots, is not functional in dogs and their wild relatives, wolves and coyotes.

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    Violeta Muñoz-Fuentes

    Full Text Available Meiotic recombination is a fundamental process needed for the correct segregation of chromosomes during meiosis in sexually reproducing organisms. In humans, 80% of crossovers are estimated to occur at specific areas of the genome called recombination hotspots. Recently, a protein called PRDM9 was identified as a major player in determining the location of genome-wide meiotic recombination hotspots in humans and mice. The origin of this protein seems to be ancient in evolutionary time, as reflected by its fairly conserved structure in lineages that diverged over 700 million years ago. Despite its important role, there are many animal groups in which Prdm9 is absent (e.g. birds, reptiles, amphibians, diptera and it has been suggested to have disruptive mutations and thus to be a pseudogene in dogs. Because of the dog's history through domestication and artificial selection, we wanted to confirm the presence of a disrupted Prdm9 gene in dogs and determine whether this was exclusive of this species or whether it also occurred in its wild ancestor, the wolf, and in a close relative, the coyote. We sequenced the region in the dog genome that aligned to the last exon of the human Prdm9, containing the entire zinc finger domain, in 4 dogs, 17 wolves and 2 coyotes. Our results show that the three canid species possess mutations that likely make this gene non functional. Because these mutations are shared across the three species, they must have appeared prior to the split of the wolf and the coyote, millions of years ago, and are not related to domestication. In addition, our results suggest that in these three canid species recombination does not occur at hotspots or hotspot location is controlled through a mechanism yet to be determined.

  6. The SMC-5/6 Complex and the HIM-6 (BLM Helicase Synergistically Promote Meiotic Recombination Intermediate Processing and Chromosome Maturation during Caenorhabditis elegans Meiosis.

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    Ye Hong

    2016-03-01

    Full Text Available Meiotic recombination is essential for the repair of programmed double strand breaks (DSBs to generate crossovers (COs during meiosis. The efficient processing of meiotic recombination intermediates not only needs various resolvases but also requires proper meiotic chromosome structure. The Smc5/6 complex belongs to the structural maintenance of chromosome (SMC family and is closely related to cohesin and condensin. Although the Smc5/6 complex has been implicated in the processing of recombination intermediates during meiosis, it is not known how Smc5/6 controls meiotic DSB repair. Here, using Caenorhabditis elegans we show that the SMC-5/6 complex acts synergistically with HIM-6, an ortholog of the human Bloom syndrome helicase (BLM during meiotic recombination. The concerted action of the SMC-5/6 complex and HIM-6 is important for processing recombination intermediates, CO regulation and bivalent maturation. Careful examination of meiotic chromosomal morphology reveals an accumulation of inter-chromosomal bridges in smc-5; him-6 double mutants, leading to compromised chromosome segregation during meiotic cell divisions. Interestingly, we found that the lethality of smc-5; him-6 can be rescued by loss of the conserved BRCA1 ortholog BRC-1. Furthermore, the combined deletion of smc-5 and him-6 leads to an irregular distribution of condensin and to chromosome decondensation defects reminiscent of condensin depletion. Lethality conferred by condensin depletion can also be rescued by BRC-1 depletion. Our results suggest that SMC-5/6 and HIM-6 can synergistically regulate recombination intermediate metabolism and suppress ectopic recombination by controlling chromosome architecture during meiosis.

  7. The SMC-5/6 Complex and the HIM-6 (BLM) Helicase Synergistically Promote Meiotic Recombination Intermediate Processing and Chromosome Maturation during Caenorhabditis elegans Meiosis.

    Science.gov (United States)

    Hong, Ye; Sonneville, Remi; Agostinho, Ana; Meier, Bettina; Wang, Bin; Blow, J Julian; Gartner, Anton

    2016-03-01

    Meiotic recombination is essential for the repair of programmed double strand breaks (DSBs) to generate crossovers (COs) during meiosis. The efficient processing of meiotic recombination intermediates not only needs various resolvases but also requires proper meiotic chromosome structure. The Smc5/6 complex belongs to the structural maintenance of chromosome (SMC) family and is closely related to cohesin and condensin. Although the Smc5/6 complex has been implicated in the processing of recombination intermediates during meiosis, it is not known how Smc5/6 controls meiotic DSB repair. Here, using Caenorhabditis elegans we show that the SMC-5/6 complex acts synergistically with HIM-6, an ortholog of the human Bloom syndrome helicase (BLM) during meiotic recombination. The concerted action of the SMC-5/6 complex and HIM-6 is important for processing recombination intermediates, CO regulation and bivalent maturation. Careful examination of meiotic chromosomal morphology reveals an accumulation of inter-chromosomal bridges in smc-5; him-6 double mutants, leading to compromised chromosome segregation during meiotic cell divisions. Interestingly, we found that the lethality of smc-5; him-6 can be rescued by loss of the conserved BRCA1 ortholog BRC-1. Furthermore, the combined deletion of smc-5 and him-6 leads to an irregular distribution of condensin and to chromosome decondensation defects reminiscent of condensin depletion. Lethality conferred by condensin depletion can also be rescued by BRC-1 depletion. Our results suggest that SMC-5/6 and HIM-6 can synergistically regulate recombination intermediate metabolism and suppress ectopic recombination by controlling chromosome architecture during meiosis.

  8. Genome-Wide Analysis of Heteroduplex DNA in Mismatch Repair–Deficient Yeast Cells Reveals Novel Properties of Meiotic Recombination Pathways

    Science.gov (United States)

    Martini, Emmanuelle; Borde, Valérie; Legendre, Matthieu; Audic, Stéphane; Regnault, Béatrice; Soubigou, Guillaume; Dujon, Bernard; Llorente, Bertrand

    2011-01-01

    Meiotic DNA double-strand breaks (DSBs) initiate crossover (CO) recombination, which is necessary for accurate chromosome segregation, but DSBs may also repair as non-crossovers (NCOs). Multiple recombination pathways with specific intermediates are expected to lead to COs and NCOs. We revisited the mechanisms of meiotic DSB repair and the regulation of CO formation, by conducting a genome-wide analysis of strand-transfer intermediates associated with recombination events. We performed this analysis in a SK1 × S288C Saccharomyces cerevisiae hybrid lacking the mismatch repair (MMR) protein Msh2, to allow efficient detection of heteroduplex DNAs (hDNAs). First, we observed that the anti-recombinogenic activity of MMR is responsible for a 20% drop in CO number, suggesting that in MMR–proficient cells some DSBs are repaired using the sister chromatid as a template when polymorphisms are present. Second, we observed that a large fraction of NCOs were associated with trans–hDNA tracts constrained to a single chromatid. This unexpected finding is compatible with dissolution of double Holliday junctions (dHJs) during repair, and it suggests the existence of a novel control point for CO formation at the level of the dHJ intermediate, in addition to the previously described control point before the dHJ formation step. Finally, we observed that COs are associated with complex hDNA patterns, confirming that the canonical double-strand break repair model is not sufficient to explain the formation of most COs. We propose that multiple factors contribute to the complexity of recombination intermediates. These factors include repair of nicks and double-stranded gaps, template switches between non-sister and sister chromatids, and HJ branch migration. Finally, the good correlation between the strand transfer properties observed in the absence of and in the presence of Msh2 suggests that the intermediates detected in the absence of Msh2 reflect normal intermediates. PMID

  9. Genome-wide analysis of heteroduplex DNA in mismatch repair-deficient yeast cells reveals novel properties of meiotic recombination pathways.

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    Emmanuelle Martini

    2011-09-01

    Full Text Available Meiotic DNA double-strand breaks (DSBs initiate crossover (CO recombination, which is necessary for accurate chromosome segregation, but DSBs may also repair as non-crossovers (NCOs. Multiple recombination pathways with specific intermediates are expected to lead to COs and NCOs. We revisited the mechanisms of meiotic DSB repair and the regulation of CO formation, by conducting a genome-wide analysis of strand-transfer intermediates associated with recombination events. We performed this analysis in a SK1 × S288C Saccharomyces cerevisiae hybrid lacking the mismatch repair (MMR protein Msh2, to allow efficient detection of heteroduplex DNAs (hDNAs. First, we observed that the anti-recombinogenic activity of MMR is responsible for a 20% drop in CO number, suggesting that in MMR-proficient cells some DSBs are repaired using the sister chromatid as a template when polymorphisms are present. Second, we observed that a large fraction of NCOs were associated with trans-hDNA tracts constrained to a single chromatid. This unexpected finding is compatible with dissolution of double Holliday junctions (dHJs during repair, and it suggests the existence of a novel control point for CO formation at the level of the dHJ intermediate, in addition to the previously described control point before the dHJ formation step. Finally, we observed that COs are associated with complex hDNA patterns, confirming that the canonical double-strand break repair model is not sufficient to explain the formation of most COs. We propose that multiple factors contribute to the complexity of recombination intermediates. These factors include repair of nicks and double-stranded gaps, template switches between non-sister and sister chromatids, and HJ branch migration. Finally, the good correlation between the strand transfer properties observed in the absence of and in the presence of Msh2 suggests that the intermediates detected in the absence of Msh2 reflect normal intermediates.

  10. The mismatch repair protein MLH1 marks a subset of strongly interfering crossovers in tomato

    NARCIS (Netherlands)

    Lhuissier, F.G.P.; Offenberg, H.H.; Wittich, P.E.; Vischer, N.O.E.; Heyting, C.

    2007-01-01

    In most eukaryotes, the prospective chromosomal positions of meiotic crossovers are marked during meiotic prophase by protein complexes called late recombination nodules (LNs). In tomato (Solanum lycopersicum), a cytological recombination map has been constructed based on LN positions. We

  11. SOLO: a meiotic protein required for centromere cohesion, coorientation, and SMC1 localization in Drosophila melanogaster.

    Science.gov (United States)

    Yan, Rihui; Thomas, Sharon E; Tsai, Jui-He; Yamada, Yukihiro; McKee, Bruce D

    2010-02-08

    Sister chromatid cohesion is essential to maintain stable connections between homologues and sister chromatids during meiosis and to establish correct centromere orientation patterns on the meiosis I and II spindles. However, the meiotic cohesion apparatus in Drosophila melanogaster remains largely uncharacterized. We describe a novel protein, sisters on the loose (SOLO), which is essential for meiotic cohesion in Drosophila. In solo mutants, sister centromeres separate before prometaphase I, disrupting meiosis I centromere orientation and causing nondisjunction of both homologous and sister chromatids. Centromeric foci of the cohesin protein SMC1 are absent in solo mutants at all meiotic stages. SOLO and SMC1 colocalize to meiotic centromeres from early prophase I until anaphase II in wild-type males, but both proteins disappear prematurely at anaphase I in mutants for mei-S332, which encodes the Drosophila homologue of the cohesin protector protein shugoshin. The solo mutant phenotypes and the localization patterns of SOLO and SMC1 indicate that they function together to maintain sister chromatid cohesion in Drosophila meiosis.

  12. The C. elegans DSB-2 protein reveals a regulatory network that controls competence for meiotic DSB formation and promotes crossover assurance.

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    Simona Rosu

    Full Text Available For most organisms, chromosome segregation during meiosis relies on deliberate induction of DNA double-strand breaks (DSBs and repair of a subset of these DSBs as inter-homolog crossovers (COs. However, timing and levels of DSB formation must be tightly controlled to avoid jeopardizing genome integrity. Here we identify the DSB-2 protein, which is required for efficient DSB formation during C. elegans meiosis but is dispensable for later steps of meiotic recombination. DSB-2 localizes to chromatin during the time of DSB formation, and its disappearance coincides with a decline in RAD-51 foci marking early recombination intermediates and precedes appearance of COSA-1 foci marking CO-designated sites. These and other data suggest that DSB-2 and its paralog DSB-1 promote competence for DSB formation. Further, immunofluorescence analyses of wild-type gonads and various meiotic mutants reveal that association of DSB-2 with chromatin is coordinated with multiple distinct aspects of the meiotic program, including the phosphorylation state of nuclear envelope protein SUN-1 and dependence on RAD-50 to load the RAD-51 recombinase at DSB sites. Moreover, association of DSB-2 with chromatin is prolonged in mutants impaired for either DSB formation or formation of downstream CO intermediates. These and other data suggest that association of DSB-2 with chromatin is an indicator of competence for DSB formation, and that cells respond to a deficit of CO-competent recombination intermediates by prolonging the DSB-competent state. In the context of this model, we propose that formation of sufficient CO-competent intermediates engages a negative feedback response that leads to cessation of DSB formation as part of a major coordinated transition in meiotic prophase progression. The proposed negative feedback regulation of DSB formation simultaneously (1 ensures that sufficient DSBs are made to guarantee CO formation and (2 prevents excessive DSB levels that could

  13. Distinct DNA-binding surfaces in the ATPase and linker domains of MutLγ determine its substrate specificities and exert separable functions in meiotic recombination and mismatch repair.

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    Corentin Claeys Bouuaert

    2017-05-01

    Full Text Available Mlh1-Mlh3 (MutLγ is a mismatch repair factor with a central role in formation of meiotic crossovers, presumably through resolution of double Holliday junctions. MutLγ has DNA-binding, nuclease, and ATPase activities, but how these relate to one another and to in vivo functions are unclear. Here, we combine biochemical and genetic analyses to characterize Saccharomyces cerevisiae MutLγ. Limited proteolysis and atomic force microscopy showed that purified recombinant MutLγ undergoes ATP-driven conformational changes. In vitro, MutLγ displayed separable DNA-binding activities toward Holliday junctions (HJ and, surprisingly, single-stranded DNA (ssDNA, which was not predicted from current models. MutLγ bound DNA cooperatively, could bind multiple substrates simultaneously, and formed higher-order complexes. FeBABE hydroxyl radical footprinting indicated that the DNA-binding interfaces of MutLγ for ssDNA and HJ substrates only partially overlap. Most contacts with HJ substrates were located in the linker regions of MutLγ, whereas ssDNA contacts mapped within linker regions as well as the N-terminal ATPase domains. Using yeast genetic assays for mismatch repair and meiotic recombination, we found that mutations within different DNA-binding surfaces exert separable effects in vivo. For example, mutations within the Mlh1 linker conferred little or no meiotic phenotype but led to mismatch repair deficiency. Interestingly, mutations in the N-terminal domain of Mlh1 caused a stronger meiotic defect than mlh1Δ, suggesting that the mutant proteins retain an activity that interferes with alternative recombination pathways. Furthermore, mlh3Δ caused more chromosome missegregation than mlh1Δ, whereas mlh1Δ but not mlh3Δ partially alleviated meiotic defects of msh5Δ mutants. These findings illustrate functional differences between Mlh1 and Mlh3 during meiosis and suggest that their absence impinges on chromosome segregation not only via reduced

  14. Improving recombinant protein purification yield

    Science.gov (United States)

    Production of adequate amounts of recombinant proteins is essential for antibody production, biochemical activity study, and structural determination during the post-genomic era. It’s technologically challenging and a limiting factor for tung oil research because analytical reagents such as high qua...

  15. The Fanconi anemia ortholog FANCM ensures ordered homologous recombination in both somatic and meiotic cells in Arabidopsis.

    Science.gov (United States)

    Knoll, Alexander; Higgins, James D; Seeliger, Katharina; Reha, Sarah J; Dangel, Natalie J; Bauknecht, Markus; Schröpfer, Susan; Franklin, F Christopher H; Puchta, Holger

    2012-04-01

    The human hereditary disease Fanconi anemia leads to severe symptoms, including developmental defects and breakdown of the hematopoietic system. It is caused by single mutations in the FANC genes, one of which encodes the DNA translocase FANCM (for Fanconi anemia complementation group M), which is required for the repair of DNA interstrand cross-links to ensure replication progression. We identified a homolog of FANCM in Arabidopsis thaliana that is not directly involved in the repair of DNA lesions but suppresses spontaneous somatic homologous recombination via a RecQ helicase (At-RECQ4A)-independent pathway. In addition, it is required for double-strand break-induced homologous recombination. The fertility of At-fancm mutant plants is compromised. Evidence suggests that during meiosis At-FANCM acts as antirecombinase to suppress ectopic recombination-dependent chromosome interactions, but this activity is antagonized by the ZMM pathway to enable the formation of interference-sensitive crossovers and chromosome synapsis. Surprisingly, mutation of At-FANCM overcomes the sterility phenotype of an At-MutS homolog4 mutant by apparently rescuing a proportion of crossover-designated recombination intermediates via a route that is likely At-MMS and UV sensitive81 dependent. However, this is insufficient to ensure the formation of an obligate crossover. Thus, At-FANCM is not only a safeguard for genome stability in somatic cells but is an important factor in the control of meiotic crossover formation.

  16. Localization and roles of Ski8p protein in Sordaria meiosis and delineation of three mechanistically distinct steps of meiotic homolog juxtaposition.

    Science.gov (United States)

    Tessé, Sophie; Storlazzi, Aurora; Kleckner, Nancy; Gargano, Silvana; Zickler, Denise

    2003-10-28

    Ski8p is implicated in degradation of non-poly(A) and double-stranded RNA, and in meiotic DNA recombination. We have identified the Sordaria macrospora SKI8 gene. Ski8p is cytoplasmically localized in all vegetative and sexual cycle cells, and is nuclear localized, specifically in early-mid-meiotic prophase, in temporal correlation with Spo11p, the meiotic double-strand break (DSB) transesterase. Localizations of Ski8p and Spo11p are mutually interdependent. ski8 mutants exhibit defects in vegetative growth, entry into the sexual program, and sporulation. Diverse meiotic defects, also seen in spo11 mutants, are diagnostic of DSB absence, and they are restored by exogenous DSBs. These results suggest that Ski8p promotes meiotic DSB formation by acting directly within meiotic prophase chromosomes. Mutant phenotypes also divide meiotic homolog juxtaposition into three successive, mechanistically distinct steps; recognition, presynaptic alignment, and synapsis, which are distinguished by their differential dependence on DSBs.

  17. RAD51AP2, a novel vertebrate- and meiotic-specific protein, sharesa conserved RAD51-interacting C-terminal domain with RAD51AP1/PIR51

    Energy Technology Data Exchange (ETDEWEB)

    Kovalenko, Oleg V.; Wiese, Claudia; Schild, David

    2006-07-25

    Many interacting proteins regulate and/or assist the activities of RAD51, a recombinase which plays a critical role in both DNA repair and meiotic recombination. Yeast two-hybrid screening of a human testis cDNA library revealed a new protein, RAD51AP2 (RAD51 Associated Protein 2), that interacts strongly with RAD51. A full-length cDNA clone predicts a novel vertebrate specific protein of 1159 residues, and the RAD51AP2 transcript was observed only in meiotic tissue (i.e. adult testis and fetal ovary), suggesting a meiotic-specific function for RAD51AP2. In HEK293 cells the interaction of RAD51 with an ectopically-expressed recombinant large fragment of RAD51AP2 requires the C-terminal 57 residues of RAD51AP2. This RAD51-binding region shows 81% homology to the C-terminus of RAD51AP1/PIR51, an otherwise totally unrelated RAD51-binding partner that is ubiquitously expressed. Analyses using truncations and point mutations in both RAD51AP1 and RAD51AP2 demonstrate that these proteins use the same structural motif for RAD51 binding. RAD54 shares some homology with this RAD51-binding motif, but this homologous region plays only an accessory role to the adjacent main RAD51-interacting region, which has been narrowed here to 40 amino acids. A novel protein, RAD51AP2, has been discovered that interacts with RAD51 through a C-terminal motif also present in RAD51AP1.

  18. A role for Caenorhabditis elegans chromatin-associated protein HIM-17 in the proliferation vs. meiotic entry decision.

    Science.gov (United States)

    Bessler, Jessica B; Reddy, Kirthi C; Hayashi, Michiko; Hodgkin, Jonathan; Villeneuve, Anne M

    2007-04-01

    Chromatin-associated protein HIM-17 was previously shown to function in the chromosomal events of meiotic prophase. Here we report an additional role for HIM-17 in regulating the balance between germ cell proliferation and meiotic development. A cryptic function for HIM-17 in promoting meiotic entry and/or inhibiting proliferation was revealed by defects in germline organization in him-17 mutants grown at high temperature (25 degrees) and by a synthetic tumorous germline phenotype in glp-1(ar202); him-17 mutants at 15 degrees.

  19. Location of RAD51-like protein during meiotic prophase in Eimeria tenella.

    Science.gov (United States)

    Del Cacho, Emilio; Gallego, Margarita; Pagés, Marc; Barbero, José Luís; Monteagudo, Luís; Sánchez-Acedo, Caridad

    2011-05-31

    This study focuses on reporting events in Eimeria tenella oocysts from early to late prophase I in terms of RAD51 protein in association with the synaptonemal complex formed between homologous chromosomes. The aim of the study was the sequential localization of RAD51 protein, which is involved in the repair of double-strand breaks (DSBs) on the eimerian chromosomes as they synapse and desynapse. Structural Maintenance of Chromosome protein SMC3, which plays a role in synaptonemal complex formation, was labeled to identify initiation and progress of chromosome synapsis and desynapsis in parallel with the appearance and disappearance of RAD51 foci. Antibodies directed against RAD51 and cohesin subunit SMC3 proteins were labeled with either fluorescence or colloidal gold to visualize RAD51 protein foci and synaptonemal complexes. RAD51 protein localization during prophase I was studied on meiotic chromosomes spreads obtained from oocysts at different points in time after the start of sporulation. The present findings showed that foci detected with the antibody directed against RAD51 protein first appeared at the pre-leptotene stage before homologous chromosomes began pairing. Subsequently, the foci were detected in association with the lateral elements at the precise sites where synapsis were in progress. These findings lead us to suggest that in E. tenella, homologous chromosome pairing was a DSB-dependent mechanism and reinforced the participation of RAD51 protein in meiotic homology search, alignment and pairing of chromosomes. Copyright © 2010 Elsevier B.V. All rights reserved.

  20. Improving ethanol fermentation performance of Saccharomyces cerevisiae in very high-gravity fermentation through chemical mutagenesis and meiotic recombination

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Jing-Jing; Ding, Wen-Tao; Zhang, Guo-Chang; Wang, Jing-Yu [Tianjin Univ. (China). Dept. of Biochemical Engineering

    2011-08-15

    Genome shuffling is an efficient way to improve complex phenotypes under the control of multiple genes. For the improvement of strain's performance in very high-gravity (VHG) fermentation, we developed a new method of genome shuffling. A diploid ste2/ste2 strain was subjected to EMS (ethyl methanesulfonate) mutagenesis followed by meiotic recombination-mediated genome shuffling. The resulting haploid progenies were intrapopulation sterile and therefore haploid recombinant cells with improved phenotypes were directly selected under selection condition. In VHG fermentation, strain WS1D and WS5D obtained by this approach exhibited remarkably enhanced tolerance to ethanol and osmolarity, increased metabolic rate, and 15.12% and 15.59% increased ethanol yield compared to the starting strain W303D, respectively. These results verified the feasibility of the strain improvement strategy and suggested that it is a powerful and high throughput method for development of Saccharomyces cerevisiae strains with desired phenotypes that is complex and cannot be addressed with rational approaches. (orig.)

  1. Sequence requirement of the ade6-4095 meiotic recombination hotspot in Schizosaccharomyces pombe.

    Science.gov (United States)

    Foulis, Steven J; Fowler, Kyle R; Steiner, Walter W

    2018-02-01

    Homologous recombination occurs at a greatly elevated frequency in meiosis compared to mitosis and is initiated by programmed double-strand DNA breaks (DSBs). DSBs do not occur at uniform frequency throughout the genome in most organisms, but occur preferentially at a limited number of sites referred to as hotspots. The location of hotspots have been determined at nucleotide-level resolution in both the budding and fission yeasts, and while several patterns have emerged regarding preferred locations for DSB hotspots, it remains unclear why particular sites experience DSBs at much higher frequency than other sites with seemingly similar properties. Short sequence motifs, which are often sites for binding of transcription factors, are known to be responsible for a number of hotspots. In this study we identified the minimum sequence required for activity of one of such motif identified in a screen of random sequences capable of producing recombination hotspots. The experimentally determined sequence, GGTCTRGACC, closely matches the previously inferred sequence. Full hotspot activity requires an effective sequence length of 9.5 bp, whereas moderate activity requires an effective sequence length of approximately 8.2 bp and shows significant association with DSB hotspots. In combination with our previous work, this result is consistent with a large number of different sequence motifs capable of producing recombination hotspots, and supports a model in which hotspots can be rapidly regenerated by mutation as they are lost through recombination.

  2. The Meiotic Recombination Activator PRDM9 Trimethylates Both H3K36 and H3K4 at Recombination Hotspots In Vivo.

    Science.gov (United States)

    Powers, Natalie R; Parvanov, Emil D; Baker, Christopher L; Walker, Michael; Petkov, Petko M; Paigen, Kenneth

    2016-06-01

    In many mammals, including humans and mice, the zinc finger histone methyltransferase PRDM9 performs the first step in meiotic recombination by specifying the locations of hotspots, the sites of genetic recombination. PRDM9 binds to DNA at hotspots through its zinc finger domain and activates recombination by trimethylating histone H3K4 on adjacent nucleosomes through its PR/SET domain. Recently, the isolated PR/SET domain of PRDM9 was shown capable of also trimethylating H3K36 in vitro, raising the question of whether this reaction occurs in vivo during meiosis, and if so, what its function might be. Here, we show that full-length PRDM9 does trimethylate H3K36 in vivo in mouse spermatocytes. Levels of H3K4me3 and H3K36me3 are highly correlated at hotspots, but mutually exclusive elsewhere. In vitro, we find that although PRDM9 trimethylates H3K36 much more slowly than it does H3K4, PRDM9 is capable of placing both marks on the same histone molecules. In accord with these results, we also show that PRDM9 can trimethylate both K4 and K36 on the same nucleosomes in vivo, but the ratio of K4me3/K36me3 is much higher for the pair of nucleosomes adjacent to the PRDM9 binding site compared to the next pair further away. Importantly, H3K4me3/H3K36me3-double-positive nucleosomes occur only in regions of recombination: hotspots and the pseudoautosomal (PAR) region of the sex chromosomes. These double-positive nucleosomes are dramatically reduced when PRDM9 is absent, showing that this signature is PRDM9-dependent at hotspots; the residual double-positive nucleosomes most likely come from the PRDM9-independent PAR. These results, together with the fact that PRDM9 is the only known mammalian histone methyltransferase with both H3K4 and H3K36 trimethylation activity, suggest that trimethylation of H3K36 plays an important role in the recombination process. Given the known requirement of H3K36me3 for double strand break repair by homologous recombination in somatic cells, we

  3. Evidence that meiotic pairing starts at the telomeres: Molecular analysis of recombination in a family with a pericentric X chromosome inversion

    Energy Technology Data Exchange (ETDEWEB)

    Shashi, V.; Allinson, P.S.; Golden, W.L.; Kelly, T.E. [Univ. of Virginia, Charlottesville, VA (United States)

    1994-09-01

    Recent studies in yeast have shown that telomeres rather than centromeres lead in chromosome movement just prior to meiosis and may have a role in recombination. Cytological studies of meiosis in Drosophila and mice have shown that in pericentric inversion heterozygotes there is lack of loop formation, with recobmination seen only outside the inversion. In a family with Duchenne muscular dystrophy (DMD) we recognized that only affected males and carrier females had a pericentric X chromosome inversion (inv X(p11.4;q26)). Since the short arm inversion breakpoint was proximal to the DMD locus, it could not be implicated in the mutational event causing DMD. There was no history of infertility, recurrent miscarriages or liveborn unbalanced females to suggest there was recombination within the inversion. We studied 22 members over three generations to understand the pattern of meiotic recombination between the normal and the inverted X chromosome. In total, 17 meioses involving the inverted X chromosome in females were studied by cytogenetic analysis and 16 CA repeat polymorphisms along the length of the X chromosome. Results: (a) There was complete concordance between the segregation of the DMD mutation and the inverted X chromosome. (b) On DNA analysis, there was complete absence of recombination within the inverted segment. We also found no recombination at the DMD locus. Recombination was seen only at Xp22 and Xq27-28. (c) Recombination was seen in the same individual at both Xp22 and Xq27-28 without recombination otherwise. Conclusions: (1) Pericentric X inversions reduce the genetic map length of the chromosome, with the physical map length being normal. (2) Meiotic X chromosome pairing in this family is initiated at the telomeres. (3) Following telomeric pairing in pericentric X chromosome inversions, there is inhibition of recombination within the inversion and adjacent regions.

  4. Meiotic Recombination Analyses in Pigs Carrying Different Balanced Structural Chromosomal Rearrangements.

    Directory of Open Access Journals (Sweden)

    Nicolas Mary

    Full Text Available Correct pairing, synapsis and recombination between homologous chromosomes are essential for normal meiosis. All these events are strongly regulated, and our knowledge of the mechanisms involved in this regulation is increasing rapidly. Chromosomal rearrangements are known to disturb these processes. In the present paper, synapsis and recombination (number and distribution of MLH1 foci were studied in three boars (Sus scrofa domestica carrying different chromosomal rearrangements. One (T34he was heterozygote for the t(3;4(p1.3;q1.5 reciprocal translocation, one (T34ho was homozygote for that translocation, while the third (T34Inv was heterozygote for both the translocation and a pericentric inversion inv(4(p1.4;q2.3. All three boars were normal for synapsis and sperm production. This particular situation allowed us to rigorously study the impact of rearrangements on recombination. Overall, the rearrangements induced only minor modifications of the number of MLH1 foci (per spermatocyte or per chromosome and of the length of synaptonemal complexes for chromosomes 3 and 4. The distribution of MLH1 foci in T34he was comparable to that of the controls. Conversely, the distributions of MLH1 foci on chromosome 4 were strongly modified in boar T34Inv (lack of crossover in the heterosynaptic region of the quadrivalent, and crossover displaced to the chromosome extremities, and also in boar T34ho (two recombination peaks on the q-arms compared with one of higher magnitude in the controls. Analyses of boars T34he and T34Inv showed that the interference was propagated through the breakpoints. A different result was obtained for boar T34ho, in which the breakpoints (transition between SSC3 and SSC4 chromatin on the bivalents seemed to alter the transmission of the interference signal. Our results suggest that the number of crossovers and crossover interference could be regulated by partially different mechanisms.

  5. The spatial regulation of meiotic recombination hotspots: are all DSB hotspots crossover hotspots?

    Science.gov (United States)

    Serrentino, Maria-Elisabetta; Borde, Valérie

    2012-07-15

    A key step for the success of meiosis is programmed homologous recombination, during which crossovers, or exchange of chromosome arms, take place. Crossovers increase genetic diversity but their main function is to ensure accurate chromosome segregation. Defects in crossover number and position produce aneuploidies that represent the main cause of miscarriages and chromosomal abnormalities such as Down's syndrome. Recombination is initiated by the formation of programmed double strand breaks (DSBs), which occur preferentially at places called DSB hotspots. Among all DSBs generated, only a small fraction is repaired by crossover, the other being repaired by other homologous recombination pathways. Crossover maps have been generated in a number of organisms, defining crossover hotspots. With the availability of genome-wide maps of DSBs as well as the ability to measure genetically the repair outcome at several hotspots, it is becoming more and more clear that not all DSB hotspots behave the same for crossover formation, suggesting that chromosomal features distinguish different types of hotspots. Copyright © 2012. Published by Elsevier Inc.

  6. A simple model for the influence of meiotic conversion tracts on GC content.

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    Marie-Claude Marsolier-Kergoat

    Full Text Available A strong correlation between GC content and recombination rate is observed in many eukaryotes, which is thought to be due to conversion events linked to the repair of meiotic double-strand breaks. In several organisms, the length of conversion tracts has been shown to decrease exponentially with increasing distance from the sites of meiotic double-strand breaks. I show here that this behavior leads to a simple analytical model for the evolution and the equilibrium state of the GC content of sequences devoid of meiotic double-strand break sites. In the yeast Saccharomyces cerevisiae, meiotic double-strand breaks are practically excluded from protein-coding sequences. A good fit was observed between the predictions of the model and the variations of the average GC content of the third codon position (GC3 of S. cerevisiae genes. Moreover, recombination parameters that can be extracted by fitting the data to the model coincide with experimentally determined values. These results thus indicate that meiotic recombination plays an important part in determining the fluctuations of GC content in yeast coding sequences. The model also accounted for the different patterns of GC variations observed in the genes of Candida species that exhibit a variety of sexual lifestyles, and hence a wide range of meiotic recombination rates. Finally, the variations of the average GC3 content of human and chicken coding sequences could also be fitted by the model. These results suggest the existence of a widespread pattern of GC variation in eukaryotic genes due to meiotic recombination, which would imply the generality of two features of meiotic recombination: its association with GC-biased gene conversion and the quasi-exclusion of meiotic double-strand breaks from coding sequences. Moreover, the model points out to specific constraints on protein fragments encoded by exon terminal sequences, which are the most affected by the GC bias.

  7. Differences in meiotic recombination rates in childhood acute lymphoblastic leukemia at an MHC class II hotspot close to disease associated haplotypes.

    Directory of Open Access Journals (Sweden)

    Pamela Thompson

    Full Text Available Childhood Acute Lymphoblastic Leukemia (ALL is a malignant lymphoid disease of which B-cell precursor- (BCP and T-cell- (T ALL are subtypes. The role of alleles encoded by major histocompatibility loci (MHC have been examined in a number of previous studies and results indicating weak, multi-allele associations between the HLA-DPB1 locus and BCP-ALL suggested a role for immunosusceptibility and possibly infection. Two independent SNP association studies of ALL identified loci approximately 37 kb from one another and flanking a strong meiotic recombination hotspot (DNA3, adjacent to HLA-DOA and centromeric of HLA-DPB1. To determine the relationship between this observation and HLA-DPB1 associations, we constructed high density SNP haplotypes of the 316 kb region from HLA-DMB to COL11A2 in childhood ALL and controls using a UK GWAS data subset and the software PHASE. Of four haplotype blocks identified, predicted haplotypes in Block 1 (centromeric of DNA3 differed significantly between BCP-ALL and controls (P = 0.002 and in Block 4 (including HLA-DPB1 between T-ALL and controls (P = 0.049. Of specific common (>5% haplotypes in Block 1, two were less frequent in BCP-ALL, and in Block 4 a single haplotype was more frequent in T-ALL, compared to controls. Unexpectedly, we also observed apparent differences in ancestral meiotic recombination rates at DNA3, with BCP-ALL showing increased and T-ALL decreased levels compared to controls. In silico analysis using LDsplit sotware indicated that recombination rates at DNA3 are influenced by flanking loci, including SNPs identified in childhood ALL association studies. The observed differences in rates of meiotic recombination at this hotspot, and potentially others, may be a characteristic of childhood leukemia and contribute to disease susceptibility, alternatively they may reflect interactions between ALL-associated haplotypes in this region.

  8. Genetic interactions between the chromosome axis-associated protein Hop1 and homologous recombination determinants in Schizosaccharomyces pombe.

    Science.gov (United States)

    Brown, Simon David; Jarosinska, Olga Dorota; Lorenz, Alexander

    2018-03-17

    Hop1 is a component of the meiosis-specific chromosome axis and belongs to the evolutionarily conserved family of HORMA domain proteins. Hop1 and its orthologs in higher eukaryotes are a major factor in promoting double-strand DNA break formation and inter-homolog recombination. In budding yeast and mammals, they are also involved in a meiotic checkpoint kinase cascade monitoring the completion of double-strand DNA break repair. We used the fission yeast, Schizosaccharomyces pombe, which lacks a canonical synaptonemal complex to test whether Hop1 has a role beyond supporting the generation of double-strand DNA breaks and facilitating inter-homolog recombination events. We determined how mutants of homologous recombination factors genetically interact with hop1, studied the role(s) of the HORMA domain of Hop1, and characterized a bio-informatically predicted interactor of Hop1, Aho1 (SPAC688.03c). Our observations indicate that in fission yeast, Hop1 does require its HORMA domain to support wild-type levels of meiotic recombination and localization to meiotic chromatin. Furthermore, we show that hop1∆ only weakly interacts genetically with mutants of homologous recombination factors, and in fission yeast likely has no major role beyond break formation and promoting inter-homolog events. We speculate that after the evolutionary loss of the synaptonemal complex, Hop1 likely has become less important for modulating recombination outcome during meiosis in fission yeast, and that this led to a concurrent rewiring of genetic pathways controlling meiotic recombination.

  9. A Link between Meiotic Prophase Progression and CrossoverControl

    Energy Technology Data Exchange (ETDEWEB)

    Carlton, Peter M.; Farruggio, Alfonso P.; Dernburg, Abby F.

    2005-07-06

    During meiosis, most organisms ensure that homologous chromosomes undergo at least one exchange of DNA, or crossover, to link chromosomes together and accomplish proper segregation. How each chromosome receives a minimum of one crossover is unknown. During early meiosis in Caenorhabditis elegans and many other species, chromosomes adopt a polarized organization within the nucleus, which normally disappears upon completion of homolog synapsis. Mutations that impair synapsis even between a single pair of chromosomes in C. elegans delay this nuclear reorganization. We quantified this delay by developing a classification scheme for discrete stages of meiosis. Immunofluorescence localization of RAD-51 protein revealed that delayed meiotic cells also contained persistent recombination intermediates. Through genetic analysis, we found that this cytological delay in meiotic progression requires double-strand breaks and the function of the crossover-promoting heteroduplex HIM-14 (Msh4) and MSH-5. Failure of X chromosome synapsis also resulted in impaired crossover control on autosomes, which may result from greater numbers and persistence of recombination intermediates in the delayed nuclei. We conclude that maturation of recombination events on chromosomes promotes meiotic progression, and is coupled to the regulation of crossover number and placement. Our results have broad implications for the interpretation of meiotic mutants, as we have shown that asynapsis of a single chromosome pair can exert global effects on meiotic progression and recombination frequency.

  10. Female meiotic sex chromosome inactivation in chicken.

    Directory of Open Access Journals (Sweden)

    Sam Schoenmakers

    2009-05-01

    Full Text Available During meiotic prophase in male mammals, the heterologous X and Y chromosomes remain largely unsynapsed, and meiotic sex chromosome inactivation (MSCI leads to formation of the transcriptionally silenced XY body. In birds, the heterogametic sex is female, carrying Z and W chromosomes (ZW, whereas males have the homogametic ZZ constitution. During chicken oogenesis, the heterologous ZW pair reaches a state of complete heterologous synapsis, and this might enable maintenance of transcription of Z- and W chromosomal genes during meiotic prophase. Herein, we show that the ZW pair is transiently silenced, from early pachytene to early diplotene using immunocytochemistry and gene expression analyses. We propose that ZW inactivation is most likely achieved via spreading of heterochromatin from the W on the Z chromosome. Also, persistent meiotic DNA double-strand breaks (DSBs may contribute to silencing of Z. Surprisingly, gammaH2AX, a marker of DSBs, and also the earliest histone modification that is associated with XY body formation in mammalian and marsupial spermatocytes, does not cover the ZW during the synapsed stage. However, when the ZW pair starts to desynapse, a second wave of gammaH2AX accumulates on the unsynapsed regions of Z, which also show a reappearance of the DSB repair protein RAD51. This indicates that repair of meiotic DSBs on the heterologous part of Z is postponed until late pachytene/diplotene, possibly to avoid recombination with regions on the heterologously synapsed W chromosome. Two days after entering diplotene, the Z looses gammaH2AX and shows reactivation. This is the first report of meiotic sex chromosome inactivation in a species with female heterogamety, providing evidence that this mechanism is not specific to spermatogenesis. It also indicates the presence of an evolutionary force that drives meiotic sex chromosome inactivation independent of the final achievement of synapsis.

  11. The Chromatin Protein DUET/MMD1 Controls Expression of the Meiotic Gene TDM1 during Male Meiosis in Arabidopsis.

    Science.gov (United States)

    Andreuzza, Sébastien; Nishal, Bindu; Singh, Aparna; Siddiqi, Imran

    2015-09-01

    Meiosis produces haploid cells essential for sexual reproduction. In yeast, entry into meiosis activates transcription factors which trigger a transcriptional cascade that results in sequential co-expression of early, middle and late meiotic genes. However, these factors are not conserved, and the factors and regulatory mechanisms that ensure proper meiotic gene expression in multicellular eukaryotes are poorly understood. Here, we report that DUET/MMD1, a PHD finger protein essential for Arabidopsis male meiosis, functions as a transcriptional regulator in plant meiosis. We find that DUET-PHD binds H3K4me2 in vitro, and show that this interaction is critical for function during meiosis. We also show that DUET is required for proper microtubule organization during meiosis II, independently of its function in meiosis I. Remarkably, DUET protein shows stage-specific expression, confined to diplotene. We identify two genes TDM1 and JAS with critical functions in cell cycle transitions and spindle organization in male meiosis, as DUET targets, with TDM1 being a direct target. Thus, DUET is required to regulate microtubule organization and cell cycle transitions during male meiosis, and functions as a direct transcription activator of the meiotic gene TDM1. Expression profiling showed reduced expression of a subset comprising about 12% of a known set of meiosis preferred genes in the duet mutant. Our results reveal the action of DUET as a transcriptional regulator during male meiosis in plants, and suggest that transcription of meiotic genes is under stagewise control in plants as in yeast.

  12. Recombinant DNA production of spider silk proteins.

    Science.gov (United States)

    Tokareva, Olena; Michalczechen-Lacerda, Valquíria A; Rech, Elíbio L; Kaplan, David L

    2013-11-01

    Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks. © 2013 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  13. Recombinant protein blends: silk beyond natural design.

    Science.gov (United States)

    Dinjaski, Nina; Kaplan, David L

    2016-06-01

    Recombinant DNA technology and new material concepts are shaping future directions in biomaterial science for the design and production of the next-generation biomaterial platforms. Aside from conventionally used synthetic polymers, numerous natural biopolymers (e.g., silk, elastin, collagen, gelatin, alginate, cellulose, keratin, chitin, polyhydroxyalkanoates) have been investigated for properties and manipulation via bioengineering. Genetic engineering provides a path to increase structural and functional complexity of these biopolymers, and thereby expand the catalog of available biomaterials beyond that which exists in nature. In addition, the integration of experimental approaches with computational modeling to analyze sequence-structure-function relationships is starting to have an impact in the field by establishing predictive frameworks for determining material properties. Herein, we review advances in recombinant DNA-mediated protein production and functionalization approaches, with a focus on hybrids or combinations of proteins; recombinant protein blends or 'recombinamers'. We highlight the potential biomedical applications of fibrous protein recombinamers, such as Silk-Elastin Like Polypeptides (SELPs) and Silk-Bacterial Collagens (SBCs). We also discuss the possibility for the rationale design of fibrous proteins to build smart, stimuli-responsive biomaterials for diverse applications. We underline current limitations with production systems for these proteins and discuss the main trends in systems/synthetic biology that may improve recombinant fibrous protein design and production. Copyright © 2016. Published by Elsevier Ltd.

  14. A novel mouse synaptonemal complex protein is essential for loading of central element proteins, recombination, and fertility.

    Directory of Open Access Journals (Sweden)

    Sabine Schramm

    2011-05-01

    Full Text Available The synaptonemal complex (SC is a proteinaceous, meiosis-specific structure that is highly conserved in evolution. During meiosis, the SC mediates synapsis of homologous chromosomes. It is essential for proper recombination and segregation of homologous chromosomes, and therefore for genome haploidization. Mutations in human SC genes can cause infertility. In order to gain a better understanding of the process of SC assembly in a model system that would be relevant for humans, we are investigating meiosis in mice. Here, we report on a newly identified component of the murine SC, which we named SYCE3. SYCE3 is strongly conserved among mammals and localizes to the central element (CE of the SC. By generating a Syce3 knockout mouse, we found that SYCE3 is required for fertility in both sexes. Loss of SYCE3 blocks synapsis initiation and results in meiotic arrest. In the absence of SYCE3, initiation of meiotic recombination appears to be normal, but its progression is severely impaired resulting in complete absence of MLH1 foci, which are presumed markers of crossovers in wild-type meiocytes. In the process of SC assembly, SYCE3 is required downstream of transverse filament protein SYCP1, but upstream of the other previously described CE-specific proteins. We conclude that SYCE3 enables chromosome loading of the other CE-specific proteins, which in turn would promote synapsis between homologous chromosomes.

  15. Cultivating Insect Cells To Produce Recombinant Proteins

    Science.gov (United States)

    Spaulding, Glenn; Goodwin, Thomas; Prewett, Tacey; Andrews, Angela; Francis, Karen; O'Connor, Kim

    1996-01-01

    Method of producing recombinant proteins involves growth of insect cells in nutrient solution in cylindrical bioreactor rotating about cylindrical axis, oriented horizontally and infecting cells with viruses into which genes of selected type cloned. Genes in question those encoding production of desired proteins. Horizontal rotating bioreactor preferred for use in method, denoted by acronym "HARV", described in "High-Aspect-Ratio Rotating Cell-Culture Vessel" (MSC-21662).

  16. Protein Crystal Recombinant Human Insulin

    Science.gov (United States)

    1994-01-01

    The comparison of protein crystal, Recombiant Human Insulin; space-grown (left) and earth-grown (right). On STS-60, Spacehab II indicated that space-grown crystals are larger and of greater optical clarity than their earth-grown counterparts. Recombiant Human Insulin facilitates the incorporation of glucose into cells. In diabetics, there is either a decrease in or complete lack of insulin, thereby leading to several harmful complications. Principal Investigator is Larry DeLucas.

  17. Extracellular secretion of recombinant proteins

    Science.gov (United States)

    Linger, Jeffrey G.; Darzins, Aldis

    2014-07-22

    Nucleic acids encoding secretion signals, expression vectors containing the nucleic acids, and host cells containing the expression vectors are disclosed. Also disclosed are polypeptides that contain the secretion signals and methods of producing polypeptides, including methods of directing the extracellular secretion of the polypeptides. Exemplary embodiments include cellulase proteins fused to secretion signals, methods to produce and isolate these polypeptides, and methods to degrade lignocellulosic biomass.

  18. Functions and structures of eukaryotic recombination proteins

    International Nuclear Information System (INIS)

    Ogawa, Tomoko

    1994-01-01

    We have found that Rad51 and RecA Proteins form strikingly similar structures together with dsDNA and ATP. Their right handed helical nucleoprotein filaments extend the B-form DNA double helixes to 1.5 times in length and wind the helix. The similarity and uniqueness of their structures must reflect functional homologies between these proteins. Therefore, it is highly probable that similar recombination proteins are present in various organisms of different evolutional states. We have succeeded to clone RAD51 genes from human, mouse, chicken and fission yeast genes, and found that the homologues are widely distributed in eukaryotes. The HsRad51 and MmRad51 or ChRad51 proteins consist of 339 amino acids differing only by 4 or 12 amino acids, respectively, and highly homologous to both yeast proteins, but less so to Dmcl. All of these proteins are homologous to the region from residues 33 to 240 of RecA which was named ''homologous core. The homologous core is likely to be responsible for functions common for all of them, such as the formation of helical nucleoprotein filament that is considered to be involved in homologous pairing in the recombination reaction. The mouse gene is transcribed at a high level in thymus, spleen, testis, and ovary, at lower level in brain and at a further lower level in some other tissues. It is transcribed efficiently in recombination active tissues. A clear functional difference of Rad51 homologues from RecA was suggested by the failure of heterologous genes to complement the deficiency of Scrad51 mutants. This failure seems to reflect the absence of a compatible partner, such as ScRad52 protein in the case of ScRad51 protein, between different species. Thus, these discoveries play a role of the starting point to understand the fundamental gene targeting in mammalian cells and in gene therapy. (J.P.N.)

  19. Recombinant protein scaffolds for tissue engineering

    International Nuclear Information System (INIS)

    Werkmeister, Jerome A; Ramshaw, John A M

    2012-01-01

    New biological materials for tissue engineering are now being developed using common genetic engineering capabilities to clone and express a variety of genetic elements that allow cost-effective purification and scaffold fabrication from these recombinant proteins, peptides or from chimeric combinations of these. The field is limitless as long as the gene sequences are known. The utility is dependent on the ease, product yield and adaptability of these protein products to the biomedical field. The development of recombinant proteins as scaffolds, while still an emerging technology with respect to commercial products, is scientifically superior to current use of natural materials or synthetic polymer scaffolds, in terms of designing specific structures with desired degrees of biological complexities and motifs. In the field of tissue engineering, next generation scaffolds will be the key to directing appropriate tissue regeneration. The initial period of biodegradable synthetic scaffolds that provided shape and mechanical integrity, but no biological information, is phasing out. The era of protein scaffolds offers distinct advantages, particularly with the combination of powerful tools of molecular biology. These include, for example, the production of human proteins of uniform quality that are free of infectious agents and the ability to make suitable quantities of proteins that are found in low quantity or are hard to isolate from tissue. For the particular needs of tissue engineering scaffolds, fibrous proteins like collagens, elastin, silks and combinations of these offer further advantages of natural well-defined structural scaffolds as well as endless possibilities of controlling functionality by genetic manipulation. (topical review)

  20. Recombinant Amphiphilic Protein Micelles for Drug Delivery

    OpenAIRE

    Kim, Wookhyun; Xiao, Jiantao; Chaikof, Elliot L.

    2011-01-01

    Amphiphilic block polypeptides can self-assemble into a range of nanostructures in solution, including micelles and vesicles. Our group has recently described the capacity of recombinant amphiphilic diblock copolypeptides to form highly stable micelles. In this report, we demonstrate the utility of protein nanoparticles to serve as a vehicle for controlled drug delivery. Drug-loaded micelles were produced by encapsulating dipyridamole as a model hydrophobic drug with anti-inflammatory activit...

  1. Overview of the purification of recombinant proteins.

    Science.gov (United States)

    Wingfield, Paul T

    2015-04-01

    When the first version of this unit was written in 1995, protein purification of recombinant proteins was based on a variety of standard chromatographic methods and approaches, many of which were described and mentioned throughout Current Protocols in Protein Science. In the interim, there has been a shift toward an almost universal usage of the affinity or fusion tag. This may not be the case for biotechnology manufacture where affinity tags can complicate producing proteins under regulatory conditions. Regardless of the protein expression system, questions are asked as to which and how many affinity tags to use, where to attach them in the protein, and whether to engineer a self-cleavage system or simply leave them on. We will briefly address some of these issues. Also, although this overview focuses on E.coli, protein expression and purification, other commonly used expression systems are mentioned and, apart from cell-breakage methods, protein purification methods and strategies are essentially the same. Copyright © 2015 John Wiley & Sons, Inc.

  2. Recombinant Brucella abortus gene expressing immunogenic protein

    Energy Technology Data Exchange (ETDEWEB)

    Mayfield, J.E.; Tabatabai, L.B.

    1991-06-11

    This patent describes a synthetic recombinant DNA molecule containing a DNA sequence. It comprises a gene of Brucella abortus encoding an immunogenic protein having a molecular weight of approximately 31,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis under denaturing conditions, the protein having an isoelectric point around 4.9, and containing a twenty-five amino acid sequence from its amino terminal end consisting of Gln-Ala-Pro-Thr-Phe-Phe-Arg-Ile-Gly-Thr-Gly-Gly-Thr-Ala-Gly-Thr-Tyr-Tyr-Pro-Ile-Gly-Gly-Leu-Ile-Ala, wherein Gln, Ala, Pro, Thr, Phe, Arg, Ile, Gly, Tyr, and Leu, respectively, represent glutamine, alanine, proline, threonine, phenylalanine, arginine, isolecuine, glycine, tyrosine, and leucine.

  3. Assessing fluctuating evolutionary pressure in yeast and mammal evolutionary rate covariation using bioinformatics of meiotic protein genetic sequences

    Science.gov (United States)

    Dehipawala, Sunil; Nguyen, A.; Tremberger, G.; Cheung, E.; Holden, T.; Lieberman, D.; Cheung, T.

    2013-09-01

    The evolutionary rate co-variation in meiotic proteins has been reported for yeast and mammal using phylogenic branch lengths which assess retention, duplication and mutation. The bioinformatics of the corresponding DNA sequences could be classified as a diagram of fractal dimension and Shannon entropy. Results from biomedical gene research provide examples on the diagram methodology. The identification of adaptive selection using entropy marker and functional-structural diversity using fractal dimension would support a regression analysis where the coefficient of determination would serve as evolutionary pathway marker for DNA sequences and be an important component in the astrobiology community. Comparisons between biomedical genes such as EEF2 (elongation factor 2 human, mouse, etc), WDR85 in epigenetics, HAR1 in human specificity, clinical trial targeted cancer gene CD47, SIRT6 in spermatogenesis, and HLA-C in mosquito bite immunology demonstrate the diagram classification methodology. Comparisons to the SEPT4-XIAP pair in stem cell apoptosis, testesexpressed taste genes TAS1R3-GNAT3 pair, and amyloid beta APLP1-APLP2 pair with the yeast-mammal DNA sequences for meiotic proteins RAD50-MRE11 pair and NCAPD2-ICK pair have accounted for the observed fluctuating evolutionary pressure systematically. Regression with high R-sq values or a triangular-like cluster pattern for concordant pairs in co-variation among the studied species could serve as evidences for the possible location of common ancestors in the entropy-fractal dimension diagram, consistent with an example of the human-chimp common ancestor study using the FOXP2 regulated genes reported in human fetal brain study. The Deinococcus radiodurans R1 Rad-A could be viewed as an outlier in the RAD50 diagram and also in the free energy versus fractal dimension regression Cook's distance, consistent with a non-Earth source for this radiation resistant bacterium. Convergent and divergent fluctuating evolutionary

  4. Co-solute assistance in refolding of recombinant proteins | Gerami ...

    African Journals Online (AJOL)

    Prokaryotic expression system is the most widely used host for the production of recombinant proteins but inclusion body formation is a major bottleneck in the production of recombinant proteins in prokaryotic cells, especially in Escherichia coli. In vitro refolding of inclusion body into the the proteins with native ...

  5. Engineered mammalian cells for production of recombinant proteins

    DEFF Research Database (Denmark)

    2017-01-01

    The present invention relates to mammalian cells modified to provide for improved expression of a recombinant protein of interest. In particular, the invention relates to CHO cells and other host cells in which the expression of one or more endogenous secreted proteins has been disrupted, as well...... as to the preparation, identification and use of such cells in the production of recombinant proteins....

  6. Insect Larvae: A New Platform to Produce Commercial Recombinant Proteins.

    Science.gov (United States)

    Targovnik, Alexandra M; Arregui, Mariana B; Bracco, Lautaro F; Urtasun, Nicolas; Baieli, Maria F; Segura, Maria M; Simonella, Maria A; Fogar, Mariela; Wolman, Federico J; Cascone, Osvaldo; Miranda, Maria V

    2016-01-01

    In Biotechnology, the expression of recombinant proteins is a constantly growing field and different hosts are used for this purpose. Some valuable proteins cannot be produced using traditional systems. Insects from the order Lepidoptera infected with recombinant baculovirus have appeared as a good choice to express high levels of proteins, especially those with post-translational modifications. Lepidopteran insects, which are extensively distributed in the world, can be used as small protein factories, the new biofactories. Species like Bombyx mori (silkworm) have been analyzed in Asian countries to produce a great number of recombinant proteins for use in basic and applied science and industry. Many proteins expressed in this larva have been commercialized. Several recombinant proteins produced in silkworms have already been commercialized. On the other hand, species like Spodoptera frugiperda, Heliothis virescens, Rachiplusia nu, Helicoverpa zea and Trichoplusia ni are widely distributed in both the occidental world and Europe. The expression of recombinant proteins in larvae has the advantage of its low cost in comparison with insect cell cultures. A wide variety of recombinant proteins, including enzymes, hormones and vaccines, have been efficiently expressed with intact biological activity. The expression of pharmaceutically proteins, using insect larvae or cocoons, has become very attractive. This review describes the use of insect larvae as an alternative to produce commercial recombinant proteins.

  7. Improved means and methods for expressing recombinant proteins

    NARCIS (Netherlands)

    Poolman, Berend; Martinez Linares, Daniel; Gul, Nadia

    2014-01-01

    The invention relates to the field of genetic engineering and the production of recombinant proteins in microbial host cells. Provided is a method for enhanced expression of a recombinant protein of interest in a microbial host cell, comprising providing a microbial host cell wherein the function of

  8. Recombinant pinoresinol/lariciresinol reductase, recombinant dirigent protein, and methods of use

    Science.gov (United States)

    Lewis, Norman G.; Davin, Laurence B.; Dinkova-Kostova, Albena T.; Fujita, Masayuki; Gang, David R.; Sarkanen, Simo; Ford, Joshua D.

    2001-04-03

    Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.

  9. Metal binding proteins, recombinant host cells and methods

    Science.gov (United States)

    Summers, Anne O.; Caguiat, Jonathan J.

    2004-06-15

    The present disclosure provides artificial heavy metal binding proteins termed chelons by the inventors. These chelons bind cadmium and/or mercuric ions with relatively high affinity. Also disclosed are coding sequences, recombinant DNA molecules and recombinant host cells comprising those recombinant DNA molecules for expression of the chelon proteins. In the recombinant host cells or transgenic plants, the chelons can be used to bind heavy metals taken up from contaminated soil, groundwater or irrigation water and to concentrate and sequester those ions. Recombinant enteric bacteria can be used within the gastrointestinal tracts of animals or humans exposed to toxic metal ions such as mercury and/or cadmium, where the chelon recombinantly expressed in chosen in accordance with the ion to be rededicated. Alternatively, the chelons can be immobilized to solid supports to bind and concentrate heavy metals from a contaminated aqueous medium including biological fluids.

  10. Recombinant HT{sub m4} gene, protein and assays

    Science.gov (United States)

    Lim, B.; Adra, C.N.; Lelias, J.M.

    1996-09-03

    The invention relates to a recombinant DNA molecule which encodes a HT{sub m4} protein, a transformed host cell which has been stably transfected with a DNA molecule which encodes a HT{sub m4} protein and a recombinant HT{sub m4} protein. The invention also relates to a method for detecting the presence of a hereditary atopy. 2 figs.

  11. In vivo production of recombinant proteins using occluded recombinant AcMNPV-derived baculovirus vectors.

    Science.gov (United States)

    Guijarro-Pardo, Eva; Gómez-Sebastián, Silvia; Escribano, José M

    2017-12-01

    Trichoplusia ni insect larvae infected with vectors derived from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV), are an excellent alternative to insect cells cultured in conventional bioreactors to produce recombinant proteins because productivity and cost-efficiency reasons. However, there is still a lot of work to do to reduce the manual procedures commonly required in this production platform that limit its scalability. To increase the scalability of this platform technology, a current bottleneck to be circumvented in the future is the need of injection for the inoculation of larvae with polyhedrin negative baculovirus vectors (Polh-) because of the lack of oral infectivity of these viruses, which are commonly used for production in insect cell cultures. In this work we have developed a straightforward alternative to obtain orally infective vectors derived from AcMNPV and expressing recombinant proteins that can be administered to the insect larvae (Trichoplusia ni) by feeding, formulated in the insect diet. The approach developed was based on the use of a recombinant polyhedrin protein expressed by a recombinant vector (Polh+), able to co-occlude any recombinant Polh- baculovirus vector expressing a recombinant protein. A second alternative was developed by the generation of a dual vector co-expressing the recombinant polyhedrin protein and the foreign gene of interest to obtain the occluded viruses. Additionally, by the incorporation of a reporter gene into the helper Polh+ vector, it was possible the follow-up visualization of the co-occluded viruses infection in insect larvae and will help to homogenize infection conditions. By using these methodologies, the production of recombinant proteins in per os infected larvae, without manual infection procedures, was very similar in yield to that obtained by manual injection of recombinant Polh- AcMNPV-based vectors expressing the same proteins. However, further analyses will be required for a

  12. A role for the malignant brain tumour (MBT domain protein LIN-61 in DNA double-strand break repair by homologous recombination.

    Directory of Open Access Journals (Sweden)

    Nicholas M Johnson

    Full Text Available Malignant brain tumour (MBT domain proteins are transcriptional repressors that function within Polycomb complexes. Some MBT genes are tumour suppressors, but how they prevent tumourigenesis is unknown. The Caenorhabditis elegans MBT protein LIN-61 is a member of the synMuvB chromatin-remodelling proteins that control vulval development. Here we report a new role for LIN-61: it protects the genome by promoting homologous recombination (HR for the repair of DNA double-strand breaks (DSBs. lin-61 mutants manifest numerous problems associated with defective HR in germ and somatic cells but remain proficient in meiotic recombination. They are hypersensitive to ionizing radiation and interstrand crosslinks but not UV light. Using a novel reporter system that monitors repair of a defined DSB in C. elegans somatic cells, we show that LIN-61 contributes to HR. The involvement of this MBT protein in HR raises the possibility that MBT-deficient tumours may also have defective DSB repair.

  13. Recombinant human bone morphogenetic protein induces bone formation

    International Nuclear Information System (INIS)

    Wang, E.A.; Rosen, V.; D'Alessandro, J.S.; Bauduy, M.; Cordes, P.; Harada, T.; Israel, D.I.; Hewick, R.M.; Kerns, K.M.; LaPan, P.; Luxenberg, D.P.; McQuaid, D.; Moutsatsos, I.K.; Nove, J.; Wozney, J.M.

    1990-01-01

    The authors have purified and characterized active recombinant human bone morphogenetic protein (BMP) 2A. Implantation of the recombinant protein in rats showed that a single BMP can induce bone formation in vivo. A dose-response and time-course study using the rat ectopic bone formation assay revealed that implantation of 0.5-115 μg of partially purified recombinant human BMP-2A resulted in cartilage by day 7 and bone formation by day 14. The time at which bone formation occurred was dependent on the amount of BMP-2A implanted; at high doses bone formation could be observed at 5 days. The cartilage- and bone-inductive activity of the recombinant BMP-2A is histologically indistinguishable from that of bone extracts. Thus, recombinant BMP-2A has therapeutic potential to promote de novo bone formation in humans

  14. Overview of the recombinant proteins purification by affinity tags and ...

    African Journals Online (AJOL)

    From protein within isolation process which the same matter increases labor costs further and prevents application of these tags in industrial scale. Therefore proper replacement is emphasized for enzymatic removal of purification tags. Keywords: protein purification; recombinant proteins; self-cleavable tags; Intein tags; ...

  15. Production of recombinant proteins from Plasmodium falciparum in Escherichia coli.

    Science.gov (United States)

    Guerra, Ángela Patricia; Calvo, Eliana Patricia; Wasserman, Moisés; Chaparro-Olaya, Jacqueline

    2016-02-23

    The production of recombinant proteins is essential for the characterization and functional study of proteins from Plasmodium falciparum. However, the proteins of P. falciparum are among the most challenging to express, and when expression is achieved, the recombinant proteins usually fold incorrectly and lead to the formation of inclusion bodies.  To obtain and purify four recombinant proteins and to use them as antigens to produce polyclonal antibodies. The production efficiency and solubility were evaluated as the proteins were expressed in two genetically modified strains of Escherichia coli to favor the production of heterologous proteins (BL21-CodonPlus (DE3)-RIL and BL21-pG-KJE8).  The four recombinant P. falciparum proteins corresponding to partial sequences of PfMyoA (Myosin A) and PfGAP50 (gliding associated protein 50), and the complete sequences of PfMTIP (myosin tail interacting protein) and PfGAP45 (gliding associated protein 45), were produced as glutathione S-transferase-fusion proteins, purified and used for immunizing mice.  The protein expression was much more efficient in BL21-CodonPlus, the strain that contains tRNAs that are rare in wild-type E. coli, compared to the expression in BL21-pG-KJE8. In spite of the fact that BL21-pG-KJE8 overexpresses chaperones, this strain did not minimize the formation of inclusion bodies.  The use of genetically modified strains of E. coli was essential to achieve high expression levels of the four evaluated P. falciparum proteins and lead to improved solubility of two of them. The approach used here allowed us to obtain and purify four P. falciparum proteins in enough quantity to produce polyclonal antibodies in mice, and a fair amount of two pure and soluble recombinant proteins for future assays.

  16. Reactions of chicken sera to recombinant Campylobacter jejuni flagellar proteins.

    Science.gov (United States)

    Yeh, Hung-Yueh; Hiett, Kelli L; Line, John E

    2015-03-01

    Campylobacter jejuni is a Gram-negative spiral rod bacterium and is the leading but underreported bacterial food-borne pathogen that causes human campylobacteriosis worldwide. Raw or undercooked poultry products are regarded as a major source for human infection. C. jejuni flagella have been implicated in colonization and adhesion to the mucosal surface of chicken gastrointestinal tracts. Therefore, flagellar proteins would be the excellent targets for further investigation. In this report, we used the recombinant technology to generate a battery of C. jejuni flagellar proteins, which were purified by His tag affinity chromatography and determined antigenic profiles of these recombinant flagellar proteins using sera from chickens older than 6 weeks of age. The immunoblot results demonstrate that each chicken serum reacted to various numbers of recombinant flagellar proteins. Among these recombinant proteins, chicken sera reacted predominantly to the FlgE1, FlgK, FlhF, FliG and FliY proteins. These antibody screening results provide a rationale for further evaluation of these recombinant flagellar proteins as potential vaccines for chickens to improve food safety as well as investigation of host immune response to C. jejuni.

  17. Cows are not mice: the role of cyclic AMP, phosphodiesterases, and adenosine monophosphate-activated protein kinase in the maintenance of meiotic arrest in bovine oocytes.

    Science.gov (United States)

    Bilodeau-Goeseels, Sylvie

    2011-01-01

    Meiotic maturation in mammalian oocytes is initiated during fetal development, and is then arrested at the dictyate stage - possibly for several years. Oocyte meiosis resumes in preovulatory follicles in response to the lutenizing hormone (LH) surge or spontaneously when competent oocytes are removed from follicles and cultured. The mechanisms involved in meiotic arrest and resumption in bovine oocytes are not fully understood, and several studies point to important differences between oocytes from rodent and livestock species. This paper reviews earlier and contemporary studies on the effects of cAMP-elevating agents and phosphodiesterase (PDE) enzyme inhibitors on the maintenance of meiotic arrest in bovine oocytes in vitro. Contrary to results obtained with mouse oocytes, bovine oocyte meiosis is inhibited by activators of the energy sensor adenosine monophosphate-activated protein kinase (AMPK, mammalian gene PRKA), which is activated by AMP, the degradation product of cAMP. It is not clear whether or not the effects were due to AMPK activation, and they may depend on culture conditions. Evidence suggests that other signaling pathways (for example, the cGMP/nitric oxide pathway) are involved in bovine oocyte meiotic arrest, but further studies are needed to understand the interactions between the signaling pathways that lead to maturation promoting factor (MPF) being inactive or active. An improved understanding of the mechanisms involved in the control of bovine oocyte meiosis will facilitate better control of the process in vitro, resulting in increased developmental competence and increased efficiency of in vitro embryo production procedures. Copyright © 2011 Wiley Periodicals, Inc.

  18. Viral vectors for production of recombinant proteins in plants.

    Science.gov (United States)

    Lico, Chiara; Chen, Qiang; Santi, Luca

    2008-08-01

    Global demand for recombinant proteins has steadily accelerated for the last 20 years. These recombinant proteins have a wide range of important applications, including vaccines and therapeutics for human and animal health, industrial enzymes, new materials and components of novel nano-particles for various applications. The majority of recombinant proteins are produced by traditional biological "factories," that is, predominantly mammalian and microbial cell cultures along with yeast and insect cells. However, these traditional technologies cannot satisfy the increasing market demand due to prohibitive capital investment requirements. During the last two decades, plants have been under intensive investigation to provide an alternative system for cost-effective, highly scalable, and safe production of recombinant proteins. Although the genetic engineering of plant viral vectors for heterologous gene expression can be dated back to the early 1980s, recent understanding of plant virology and technical progress in molecular biology have allowed for significant improvements and fine tuning of these vectors. These breakthroughs enable the flourishing of a variety of new viral-based expression systems and their wide application by academic and industry groups. In this review, we describe the principal plant viral-based production strategies and the latest plant viral expression systems, with a particular focus on the variety of proteins produced and their applications. We will summarize the recent progress in the downstream processing of plant materials for efficient extraction and purification of recombinant proteins. (c) 2008 Wiley-Liss, Inc.

  19. Synthesis and characterization of recombinant abductin-based proteins.

    Science.gov (United States)

    Su, Renay S-C; Renner, Julie N; Liu, Julie C

    2013-12-09

    Recombinant proteins are promising tools for tissue engineering and drug delivery applications. Protein-based biomaterials have several advantages over natural and synthetic polymers, including precise control over amino acid composition and molecular weight, modular swapping of functional domains, and tunable mechanical and physical properties. In this work, we describe recombinant proteins based on abductin, an elastomeric protein that is found in the inner hinge of bivalves and functions as a coil spring to keep shells open. We illustrate, for the first time, the design, cloning, expression, and purification of a recombinant protein based on consensus abductin sequences derived from Argopecten irradians . The molecular weight of the protein was confirmed by mass spectrometry, and the protein was 94% pure. Circular dichroism studies showed that the dominant structures of abductin-based proteins were polyproline II helix structures in aqueous solution and type II β-turns in trifluoroethanol. Dynamic light scattering studies illustrated that the abductin-based proteins exhibit reversible upper critical solution temperature behavior and irreversible aggregation behavior at high temperatures. A LIVE/DEAD assay revealed that human umbilical vein endothelial cells had a viability of 98 ± 4% after being cultured for two days on the abductin-based protein. Initial cell spreading on the abductin-based protein was similar to that on bovine serum albumin. These studies thus demonstrate the potential of abductin-based proteins in tissue engineering and drug delivery applications due to the cytocompatibility and its response to temperature.

  20. Recombinant protein expression in microbial systems

    OpenAIRE

    Rosano, Germán L.; Ceccarelli, Eduardo A.

    2014-01-01

    The emergence of recombinant DNA technology during the early 70's set a revolution in molecular biology. This set of techniques was strengthened even further later on with the introduction of the polymerase chain reaction and allowed scientists to explore and understand essential life processes in an easy and straightforward way. It also marked the birth of the modern biotech industry. At that time, it was shown that eukaryotic DNA could be propagated in Escherichia coli (Morrow et al., 1974)...

  1. N-Glycosylation of Plant-produced Recombinant Proteins

    NARCIS (Netherlands)

    Bosch, H.J.; Castilho, A.; Loos, A.; Schots, A.; Steinkeller, H.

    2013-01-01

    Plants are gaining increasingly acceptance as a production platform for recombinant proteins. One reason for this is their ability to carry out posttranslational protein modifications in a similar if not identical way as mammalian cells. The capability of plants to carry out human-like complex

  2. Improving recombinant protein solubility in Escherichia coli ...

    African Journals Online (AJOL)

    user

    2010-11-22

    Nov 22, 2010 ... protein (Baneyx and Mujacic, 2004). Although ... both time-consuming and expensive (Tsumoto et al.,. 2003). Thus ... proteins (Schlieker et al., 2002). ..... Alibolandi M, Mirzahoseini H, Abedi Khalil Abad M, Azami Movahed M.

  3. Trends in recombinant protein use in animal production.

    Science.gov (United States)

    Gifre, Laia; Arís, Anna; Bach, Àlex; Garcia-Fruitós, Elena

    2017-03-04

    Recombinant technologies have made possible the production of a broad catalogue of proteins of interest, including those used for animal production. The most widely studied proteins for the animal sector are those with an important role in reproduction, feed efficiency, and health. Nowadays, mammalian cells and fungi are the preferred choice for recombinant production of hormones for reproductive purposes and fibrolytic enzymes to enhance animal performance, respectively. However, the development of low-cost products is a priority, particularly in livestock. The study of cell factories such as yeast and bacteria has notably increased in the last decades to make the new developed reproductive hormones and fibrolytic enzymes a real alternative to the marketed ones. Important efforts have also been invested to developing new recombinant strategies for prevention and therapy, including passive immunization and modulation of the immune system. This offers the possibility to reduce the use of antibiotics by controlling physiological processes and improve the efficacy of preventing infections. Thus, nowadays different recombinant fibrolytic enzymes, hormones, and therapeutic molecules with optimized properties have been successfully produced through cost-effective processes using microbial cell factories. However, despite the important achievements for reducing protein production expenses, alternative strategies to further reduce these costs are still required. In this context, it is necessary to make a giant leap towards the use of novel strategies, such as nanotechnology, that combined with recombinant technology would make recombinant molecules affordable for animal industry.

  4. AKT (protein kinase B) is implicated in meiotic maturation of porcine oocytes

    Czech Academy of Sciences Publication Activity Database

    Kalous, Jaroslav; Kubelka, Michal; Šolc, Petr; Šušor, Andrej; Motlík, Jan

    2009-01-01

    Roč. 138, - (2009), s. 645-654 ISSN 1470-1626 R&D Projects: GA ČR GA204/06/1297; GA ČR GA523/03/0857; GA ČR GA524/07/1087 Institutional research plan: CEZ:AV0Z50450515 Keywords : protein kinase * porcine oocyte * oocyte maturation Subject RIV: CE - Biochemistry Impact factor: 2.579, year: 2009

  5. High recombination rate in natural populations of Plasmodium falciparum

    NARCIS (Netherlands)

    Conway, D. J.; Roper, C.; Oduola, A. M.; Arnot, D. E.; Kremsner, P. G.; Grobusch, M. P.; Curtis, C. F.; Greenwood, B. M.

    1999-01-01

    Malaria parasites are sexually reproducing protozoa, although the extent of effective meiotic recombination in natural populations has been debated. If meiotic recombination occurs frequently, compared with point mutation and mitotic rearrangement, linkage disequilibrium between polymorphic sites is

  6. Manufacturing of recombinant therapeutic proteins in microbial systems.

    Science.gov (United States)

    Graumann, Klaus; Premstaller, Andreas

    2006-02-01

    Recombinant therapeutic proteins have gained enormous importance for clinical applications. The first recombinant products have been produced in E. coli more than 20 years ago. Although with the advent of antibody-based therapeutics mammalian expression systems have experienced a major boost, microbial expression systems continue to be widely used in industry. Their intrinsic advantages, such as rapid growth, high yields and ease of manipulation, make them the premier choice for expression of non-glycosylated peptides and proteins. Innovative product classes such as antibody fragments or alternative binding molecules will further expand the use of microbial systems. Even more, novel, engineered production hosts and integrated technology platforms hold enormous potential for future applications. This review summarizes current applications and trends for development, production and analytical characterization of recombinant therapeutic proteins in microbial systems.

  7. Structural analysis of recombinant human protein QM

    International Nuclear Information System (INIS)

    Gualberto, D.C.H.; Fernandes, J.L.; Silva, F.S.; Saraiva, K.W.; Affonso, R.; Pereira, L.M.; Silva, I.D.C.G.

    2012-01-01

    Full text: The ribosomal protein QM belongs to a family of ribosomal proteins, which is highly conserved from yeast to humans. The presence of the QM protein is necessary for joining the 60S and 40S subunits in a late step of the initiation of mRNA translation. Although the exact extra-ribosomal functions of QM are not yet fully understood, it has been identified as a putative tumor suppressor. This protein was reported to interact with the transcription factor c-Jun and thereby prevent c-Jun actives genes of the cellular growth. In this study, the human QM protein was expressed in bacterial system, in the soluble form and this structure was analyzed by Circular Dichroism and Fluorescence. The results of Circular Dichroism showed that this protein has less alpha helix than beta sheet, as described in the literature. QM protein does not contain a leucine zipper region; however the ion zinc is necessary for binding of QM to c-Jun. Then we analyzed the relationship between the removal of zinc ions and folding of protein. Preliminary results obtained by the technique Fluorescence showed a gradual increase in fluorescence with the addition of increasing concentration of EDTA. This suggests that the zinc is important in the tertiary structure of the protein. More studies are being made for better understand these results. (author)

  8. Genome engineering for improved recombinant protein expression in Escherichia coli.

    Science.gov (United States)

    Mahalik, Shubhashree; Sharma, Ashish K; Mukherjee, Krishna J

    2014-12-19

    A metabolic engineering perspective which views recombinant protein expression as a multistep pathway allows us to move beyond vector design and identify the downstream rate limiting steps in expression. In E.coli these are typically at the translational level and the supply of precursors in the form of energy, amino acids and nucleotides. Further recombinant protein production triggers a global cellular stress response which feedback inhibits both growth and product formation. Countering this requires a system level analysis followed by a rational host cell engineering to sustain expression for longer time periods. Another strategy to increase protein yields could be to divert the metabolic flux away from biomass formation and towards recombinant protein production. This would require a growth stoppage mechanism which does not affect the metabolic activity of the cell or the transcriptional or translational efficiencies. Finally cells have to be designed for efficient export to prevent buildup of proteins inside the cytoplasm and also simplify downstream processing. The rational and the high throughput strategies that can be used for the construction of such improved host cell platforms for recombinant protein expression is the focus of this review.

  9. Yeast synthetic biology for the production of recombinant therapeutic proteins.

    Science.gov (United States)

    Kim, Hyunah; Yoo, Su Jin; Kang, Hyun Ah

    2015-02-01

    The production of recombinant therapeutic proteins is one of the fast-growing areas of molecular medicine and currently plays an important role in treatment of several diseases. Yeasts are unicellular eukaryotic microbial host cells that offer unique advantages in producing biopharmaceutical proteins. Yeasts are capable of robust growth on simple media, readily accommodate genetic modifications, and incorporate typical eukaryotic post-translational modifications. Saccharomyces cerevisiae is a traditional baker's yeast that has been used as a major host for the production of biopharmaceuticals; however, several nonconventional yeast species including Hansenula polymorpha, Pichia pastoris, and Yarrowia lipolytica have gained increasing attention as alternative hosts for the industrial production of recombinant proteins. In this review, we address the established and emerging genetic tools and host strains suitable for recombinant protein production in various yeast expression systems, particularly focusing on current efforts toward synthetic biology approaches in developing yeast cell factories for the production of therapeutic recombinant proteins. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

  10. Gene Delivery into Plant Cells for Recombinant Protein Production

    Directory of Open Access Journals (Sweden)

    Qiang Chen

    2015-01-01

    Full Text Available Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration in Nicotiana and non-Nicotiana plant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications.

  11. Carbon source feeding strategies for recombinant protein ...

    African Journals Online (AJOL)

    USER

    2010-04-12

    Apr 12, 2010 ... protein expression with the influence of the carbon source feeding ... in the culture media, increasing the peroxisomes numbers ...... source, temperature, pH, O2, methanol feeding strategy) ..... Catabolite Inactivation in Yeast.

  12. Cellular Reprogramming Employing Recombinant Sox2 Protein

    Directory of Open Access Journals (Sweden)

    Marc Thier

    2012-01-01

    Full Text Available Induced pluripotent stem (iPS cells represent an attractive option for the derivation of patient-specific pluripotent cells for cell replacement therapies as well as disease modeling. To become clinically meaningful, safe iPS cells need to be generated exhibiting no permanent genetic modifications that are caused by viral integrations of the reprogramming transgenes. Recently, various experimental strategies have been applied to accomplish transgene-free derivation of iPS cells, including the use of nonintegrating viruses, episomal expression, or excision of transgenes after reprogramming by site-specific recombinases or transposases. A straightforward approach to induce reprogramming factors is the direct delivery of either synthetic mRNA or biologically active proteins. We previously reported the generation of cell-permeant versions of Oct4 (Oct4-TAT and Sox2 (Sox2-TAT proteins and showed that Oct4-TAT is reprogramming-competent, that is, it can substitute for Oct4-encoding virus. Here, we explore conditions for enhanced Sox2-TAT protein stabilization and functional delivery into somatic cells. We show that cell-permeant Sox2 protein can be stabilized by lipid-rich albumin supplements in serum replacement or low-serum-supplemented media. Employing optimized conditions for protein delivery, we demonstrate that Sox2-TAT protein is able to substitute for viral Sox2. Sox2-piPS cells express pluripotency-associated markers and differentiate into all three germ layers.

  13. Expression and purification of recombinant polyomavirus VP2 protein and its interactions with polyomavirus proteins

    Science.gov (United States)

    Cai, X.; Chang, D.; Rottinghaus, S.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Recombinant polyomavirus VP2 protein was expressed in Escherichia coli (RK1448), using the recombinant expression system pFPYV2. Recombinant VP2 was purified to near homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroelution, and Extracti-Gel chromatography. Polyclonal serum to this protein which reacted specifically with recombinant VP2 as well as polyomavirus virion VP2 and VP3 on Western blots (immunoblots) was produced. Purified VP2 was used to establish an in vitro protein-protein interaction assay with polyomavirus structural proteins and purified recombinant VP1. Recombinant VP2 interacted with recombinant VP1, virion VP1, and the four virion histones. Recombinant VP1 coimmunoprecipitated with recombinant VP2 or truncated VP2 (delta C12VP2), which lacked the carboxy-terminal 12 amino acids. These experiments confirmed the interaction between VP1 and VP2 and revealed that the carboxyterminal 12 amino acids of VP2 and VP3 were not necessary for formation of this interaction. In vivo VP1-VP2 interaction study accomplished by cotransfection of COS-7 cells with VP2 and truncated VP1 (delta N11VP1) lacking the nuclear localization signal demonstrated that VP2 was capable of translocating delta N11VP1 into the nucleus. These studies suggest that complexes of VP1 and VP2 may be formed in the cytoplasm and cotransported to the nucleus for virion assembly to occur.

  14. Isolation of recombinant cysteine dioxygenase protein from Trichophyton mentagrophytes

    Czech Academy of Sciences Publication Activity Database

    Kašperová, A.; Kunert, J.; Horynová, M.; Weigl, E.; Sebela, M.; Lenobel, René; Raška, M.

    2011-01-01

    Roč. 54, č. 5 (2011), E456-E462 ISSN 0933-7407 R&D Projects: GA ČR GA301/08/1649 Institutional research plan: CEZ:AV0Z50380511 Keywords : Cysteine dioxygenase * dermatophytes * recombinant protein * keratinolytic fungi * cDNA Subject RIV: CE - Biochemistry Impact factor: 2.247, year: 2011

  15. Rec2 Interplay with both Brh2 and Rad51 Balances Recombinational Repair in Ustilago maydis

    DEFF Research Database (Denmark)

    Kojic, M.; Zhou, Q.; Lisby, M.

    2006-01-01

    and allelic recombination are elevated. The Dss1-independent Brh2-RPA70 fusion protein is also active in restoring radiation sensitivity of rec2 but is hyperactive to an extreme degree in allelic recombination and in suppressing the meiotic block of rec2. However, the high frequency of chromosome...

  16. Step-wise refolding of recombinant proteins.

    Science.gov (United States)

    Tsumoto, Kouhei; Arakawa, Tsutomu; Chen, Linda

    2010-04-01

    Protein refolding is still on trial-and-error basis. Here we describe step-wise dialysis refolding, in which denaturant concentration is altered in step-wise fashion. This technology controls the folding pathway by adjusting the concentrations of the denaturant and other solvent additives to induce sequential folding or disulfide formation.

  17. The fission yeast RNA binding protein Mmi1 regulates meiotic genes by controlling intron specific splicing and polyadenylation coupled RNA turnover.

    Directory of Open Access Journals (Sweden)

    Huei-Mei Chen

    Full Text Available The polyA tails of mRNAs are monitored by the exosome as a quality control mechanism. We find that fission yeast, Schizosaccharomyces pombe, adopts this RNA quality control mechanism to regulate a group of 30 or more meiotic genes at the level of both splicing and RNA turnover. In vegetative cells the RNA binding protein Mmi1 binds to the primary transcripts of these genes. We find the novel motif U(U/C/GAAAC highly over-represented in targets of Mmi1. Mmi1 can specifically regulate the splicing of particular introns in a transcript: it inhibits the splicing of introns that are in the vicinity of putative Mmi1 binding sites, while allowing the splicing of other introns that are far from such sites. In addition, binding of Mmi1, particularly near the 3' end, alters 3' processing to promote extremely long polyA tails of up to a kilobase. The hyperadenylated transcripts are then targeted for degradation by the nuclear exonuclease Rrp6. The nuclear polyA binding protein Pab2 assists this hyperadenylation-mediated RNA decay. Rrp6 also targets other hyperadenylated transcripts, which become hyperadenylated in an unknown, but Mmi1-independent way. Thus, hyperadenylation may be a general signal for RNA degradation. In addition, binding of Mmi1 can affect the efficiency of 3' cleavage. Inactivation of Mmi1 in meiosis allows meiotic expression, through splicing and RNA stabilization, of at least 29 target genes, which are apparently constitutively transcribed.

  18. Divergence, recombination and retention of functionality during protein evolution

    Directory of Open Access Journals (Sweden)

    Xu Yanlong O

    2005-09-01

    Full Text Available Abstract We have only a vague idea of precisely how protein sequences evolve in the context of protein structure and function. This is primarily because structural and functional contexts are not easily predictable from the primary sequence, and evaluating patterns of evolution at individual residue positions is also difficult. As a result of increasing biodiversity in genomics studies, progress is being made in detecting context-dependent variation in substitution processes, but it remains unclear exactly what context-dependent patterns we should be looking for. To address this, we have been simulating protein evolution in the context of structure and function using lattice models of proteins and ligands (or substrates. These simulations include thermodynamic features of protein stability and population dynamics. We refer to this approach as 'ab initio evolution' to emphasise the fact that the equilibrium details of fitness distributions arise from the physical principles of the system and not from any preconceived notions or arbitrary mathematical distributions. Here, we present results on the retention of functionality in homologous recombinants following population divergence. A central result is that protein structure characteristics can strongly influence recombinant functionality. Exceptional structures with many sequence options evolve quickly and tend to retain functionality -- even in highly diverged recombinants. By contrast, the more common structures with fewer sequence options evolve more slowly, but the fitness of recombinants drops off rapidly as homologous proteins diverge. These results have implications for understanding viral evolution, speciation and directed evolutionary experiments. Our analysis of the divergence process can also guide improved methods for accurately approximating folding probabilities in more complex but realistic systems.

  19. Influenza vaccines: from whole virus preparations to recombinant protein technology.

    Science.gov (United States)

    Huber, Victor C

    2014-01-01

    Vaccination against influenza represents our most effective form of prevention. Historical approaches toward vaccine creation and production have yielded highly effective vaccines that are safe and immunogenic. Despite their effectiveness, these historical approaches do not allow for the incorporation of changes into the vaccine in a timely manner. In 2013, a recombinant protein-based vaccine that induces immunity toward the influenza virus hemagglutinin was approved for use in the USA. This vaccine represents the first approved vaccine formulation that does not require an influenza virus intermediate for production. This review presents a brief history of influenza vaccines, with insight into the potential future application of vaccines generated using recombinant technology.

  20. Antibodies to a recombinant glutamate-rich Plasmodium falciparum protein

    DEFF Research Database (Denmark)

    Hogh, B; Petersen, E; Dziegiel, Morten Hanefeld

    1992-01-01

    A Plasmodium falciparum antigen gene coding for a 220-kD glutamate-rich protein (GLURP) has been cloned, and the 783 C-terminal amino acids of this protein (GLURP489-1271) have been expressed as a beta-galactosidase fusion protein in Escherichia coli. The encoded 783 amino acid residues contain two...... areas of repeated amino acid sequences. Antibodies against recombinant GLURP489-1271, as well as against a synthetic peptide corresponding to GLURP899-916, and against a synthetic peptide representing the major glutamate rich repeat sequence from the P. falciparum ring erythrocyte surface antigen (Pf155...... between the anti-GLURP489-1271 and anti-(EENV)6 antibody responses. The data provide indirect evidence for a protective role of antibodies reacting with recombinant GLURP489-1271 as well as with the synthetic peptide (EENV)6 from the Pf155/RESA....

  1. Recombinant protein production from stable mammalian cell lines and pools.

    Science.gov (United States)

    Hacker, David L; Balasubramanian, Sowmya

    2016-06-01

    We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. The cohesion protein SOLO associates with SMC1 and is required for synapsis, recombination, homolog bias and cohesion and pairing of centromeres in Drosophila Meiosis.

    Science.gov (United States)

    Yan, Rihui; McKee, Bruce D

    2013-01-01

    Cohesion between sister chromatids is mediated by cohesin and is essential for proper meiotic segregation of both sister chromatids and homologs. solo encodes a Drosophila meiosis-specific cohesion protein with no apparent sequence homology to cohesins that is required in male meiosis for centromere cohesion, proper orientation of sister centromeres and centromere enrichment of the cohesin subunit SMC1. In this study, we show that solo is involved in multiple aspects of meiosis in female Drosophila. Null mutations in solo caused the following phenotypes: 1) high frequencies of homolog and sister chromatid nondisjunction (NDJ) and sharply reduced frequencies of homolog exchange; 2) reduced transmission of a ring-X chromosome, an indicator of elevated frequencies of sister chromatid exchange (SCE); 3) premature loss of centromere pairing and cohesion during prophase I, as indicated by elevated foci counts of the centromere protein CID; 4) instability of the lateral elements (LE)s and central regions of synaptonemal complexes (SCs), as indicated by fragmented and spotty staining of the chromosome core/LE component SMC1 and the transverse filament protein C(3)G, respectively, at all stages of pachytene. SOLO and SMC1 are both enriched on centromeres throughout prophase I, co-align along the lateral elements of SCs and reciprocally co-immunoprecipitate from ovarian protein extracts. Our studies demonstrate that SOLO is closely associated with meiotic cohesin and required both for enrichment of cohesin on centromeres and stable assembly of cohesin into chromosome cores. These events underlie and are required for stable cohesion of centromeres, synapsis of homologous chromosomes, and a recombination mechanism that suppresses SCE to preferentially generate homolog crossovers (homolog bias). We propose that SOLO is a subunit of a specialized meiotic cohesin complex that mediates both centromeric and axial arm cohesion and promotes homolog bias as a component of chromosome

  3. A Bacillus megaterium System for the Production of Recombinant Proteins and Protein Complexes.

    Science.gov (United States)

    Biedendieck, Rebekka

    2016-01-01

    For many years the Gram-positive bacterium Bacillus megaterium has been used for the production and secretion of recombinant proteins. For this purpose it was systematically optimized. Plasmids with different inducible promoter systems, with different compatible origins, with small tags for protein purification and with various specific signals for protein secretion were combined with genetically improved host strains. Finally, the development of appropriate cultivation conditions for the production strains established this organism as a bacterial cell factory even for large proteins. Along with the overproduction of individual proteins the organism is now also used for the simultaneous coproduction of up to 14 recombinant proteins, multiple subsequently interacting or forming protein complexes. Some of these recombinant strains are successfully used for bioconversion or the biosynthesis of valuable components including vitamins. The titers in the g per liter scale for the intra- and extracellular recombinant protein production prove the high potential of B. megaterium for industrial applications. It is currently further enhanced for the production of recombinant proteins and multi-subunit protein complexes using directed genetic engineering approaches based on transcriptome, proteome, metabolome and fluxome data.

  4. The Pch2 AAA+ ATPase promotes phosphorylation of the Hop1 meiotic checkpoint adaptor in response to synaptonemal complex defects.

    Science.gov (United States)

    Herruzo, Esther; Ontoso, David; González-Arranz, Sara; Cavero, Santiago; Lechuga, Ana; San-Segundo, Pedro A

    2016-09-19

    Meiotic cells possess surveillance mechanisms that monitor critical events such as recombination and chromosome synapsis. Meiotic defects resulting from the absence of the synaptonemal complex component Zip1 activate a meiosis-specific checkpoint network resulting in delayed or arrested meiotic progression. Pch2 is an evolutionarily conserved AAA+ ATPase required for the checkpoint-induced meiotic block in the zip1 mutant, where Pch2 is only detectable at the ribosomal DNA array (nucleolus). We describe here that high levels of the Hop1 protein, a checkpoint adaptor that localizes to chromosome axes, suppress the checkpoint defect of a zip1 pch2 mutant restoring Mek1 activity and meiotic cell cycle delay. We demonstrate that the critical role of Pch2 in this synapsis checkpoint is to sustain Mec1-dependent phosphorylation of Hop1 at threonine 318. We also show that the ATPase activity of Pch2 is essential for its checkpoint function and that ATP binding to Pch2 is required for its localization. Previous work has shown that Pch2 negatively regulates Hop1 chromosome abundance during unchallenged meiosis. Based on our results, we propose that, under checkpoint-inducing conditions, Pch2 also possesses a positive action on Hop1 promoting its phosphorylation and its proper distribution on unsynapsed chromosome axes. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Combinatorial regulation of meiotic holliday junction resolution in C. elegans by HIM-6 (BLM) helicase, SLX-4, and the SLX-1, MUS-81 and XPF-1 nucleases.

    Science.gov (United States)

    Agostinho, Ana; Meier, Bettina; Sonneville, Remi; Jagut, Marlène; Woglar, Alexander; Blow, Julian; Jantsch, Verena; Gartner, Anton

    2013-01-01

    Holliday junctions (HJs) are cruciform DNA structures that are created during recombination events. It is a matter of considerable importance to determine the resolvase(s) that promote resolution of these structures. We previously reported that C. elegans GEN-1 is a symmetrically cleaving HJ resolving enzyme required for recombinational repair, but we could not find an overt role in meiotic recombination. Here we identify C. elegans proteins involved in resolving meiotic HJs. We found no evidence for a redundant meiotic function of GEN-1. In contrast, we discovered two redundant HJ resolution pathways likely coordinated by the SLX-4 scaffold protein and also involving the HIM-6/BLM helicase. SLX-4 associates with the SLX-1, MUS-81 and XPF-1 nucleases and has been implicated in meiotic recombination in C. elegans. We found that C. elegans [mus-81; xpf-1], [slx-1; xpf-1], [mus-81; him-6] and [slx-1; him-6] double mutants showed a similar reduction in survival rates as slx-4. Analysis of meiotic diakinesis chromosomes revealed a distinct phenotype in these double mutants. Instead of wild-type bivalent chromosomes, pairs of "univalents" linked by chromatin bridges occur. These linkages depend on the conserved meiosis-specific transesterase SPO-11 and can be restored by ionizing radiation, suggesting that they represent unresolved meiotic HJs. This suggests the existence of two major resolvase activities, one provided by XPF-1 and HIM-6, the other by SLX-1 and MUS-81. In all double mutants crossover (CO) recombination is reduced but not abolished, indicative of further redundancy in meiotic HJ resolution. Real time imaging revealed extensive chromatin bridges during the first meiotic division that appear to be eventually resolved in meiosis II, suggesting back-up resolution activities acting at or after anaphase I. We also show that in HJ resolution mutants, the restructuring of chromosome arms distal and proximal to the CO still occurs, suggesting that CO initiation

  6. Mutations in Caenorhabditis elegans him-19 show meiotic defects that worsen with age.

    Science.gov (United States)

    Tang, Lois; Machacek, Thomas; Mamnun, Yasmine M; Penkner, Alexandra; Gloggnitzer, Jiradet; Wegrostek, Christina; Konrat, Robert; Jantsch, Michael F; Loidl, Josef; Jantsch, Verena

    2010-03-15

    From a screen for meiotic Caenorhabditis elegans mutants based on high incidence of males, we identified a novel gene, him-19, with multiple functions in prophase of meiosis I. Mutant him-19(jf6) animals show a reduction in pairing of homologous chromosomes and subsequent bivalent formation. Consistently, synaptonemal complex formation is spatially restricted and possibly involves nonhomologous chromosomes. Also, foci of the recombination protein RAD-51 occur delayed or cease altogether. Ultimately, mutation of him-19 leads to chromosome missegregation and reduced offspring viability. The observed defects suggest that HIM-19 is important for both homology recognition and formation of meiotic DNA double-strand breaks. It therefore seems to be engaged in an early meiotic event, resembling in this respect the regulator kinase CHK-2. Most astonishingly, him-19(jf6) hermaphrodites display worsening of phenotypes with increasing age, whereas defects are more severe in female than in male meiosis. This finding is consistent with depletion of a him-19-dependent factor during the production of oocytes. Further characterization of him-19 could contribute to our understanding of age-dependent meiotic defects in humans.

  7. Targeted in vivo inhibition of specific protein-protein interactions using recombinant antibodies.

    Directory of Open Access Journals (Sweden)

    Matej Zábrady

    Full Text Available With the growing availability of genomic sequence information, there is an increasing need for gene function analysis. Antibody-mediated "silencing" represents an intriguing alternative for the precise inhibition of a particular function of biomolecules. Here, we describe a method for selecting recombinant antibodies with a specific purpose in mind, which is to inhibit intrinsic protein-protein interactions in the cytosol of plant cells. Experimental procedures were designed for conveniently evaluating desired properties of recombinant antibodies in consecutive steps. Our selection method was successfully used to develop a recombinant antibody inhibiting the interaction of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 3 with such of its upstream interaction partners as the receiver domain of CYTOKININ INDEPENDENT HISTIDINE KINASE 1. The specific down-regulation of the cytokinin signaling pathway in vivo demonstrates the validity of our approach. This selection method can serve as a prototype for developing unique recombinant antibodies able to interfere with virtually any biomolecule in the living cell.

  8. Potency assay design for adjuvanted recombinant proteins as malaria vaccines.

    Science.gov (United States)

    Giersing, Birgitte K; Dubovsky, Filip; Saul, Allan; Denamur, Francoise; Minor, Philip; Meade, Bruce

    2006-05-15

    Many licensed vaccines are composed of live, attenuated or inactivated whole-cell microorganisms, or they comprise purified components from whole-cell extracts or culture supernatants. For some diseases, pathology is fairly well understood, and there may be known correlates of protection that provide obvious parameters for assessment of vaccine potency. However, this is not always the case, and some effective vaccines are routinely used even though the mechanisms or correlates of protection are unknown. Some more modern vaccine approaches employ purified recombinant proteins, based on molecules that appear on the surface of the pathogen. This is one of the strategies that has been adopted in the quest to develop a malaria vaccine. Use of these parasite antigens as vaccine candidates is supported by substantial epidemiological data, and some have demonstrated the ability to elicit protective responses in animal models of malaria infection. However, there is as yet no immunological correlate of protection and no functional assays or animal models that have demonstrated the ability to predict efficacy in humans. There is little precedence for the most appropriate and practical method for assessing potency of vaccines based on these recombinant molecules for malaria vaccines. This is likely because the majority of malaria vaccine candidates have only recently entered clinical evaluation. The PATH Malaria Vaccine Initiative (MVI) convened a panel with expertise in potency assay design from industry, governmental institutions, and regulatory bodies to discuss and review the rationale, available methods, and best approaches for assessing the potency of recombinant proteins, specifically for their use as malarial vaccines. The aim of this meeting was to produce a discussion document on the practical potency assessment of recombinant protein malaria vaccines, focusing on early phase potency assay development.

  9. Impact of protein uptake and degradation on recombinant protein secretion in yeast

    DEFF Research Database (Denmark)

    Tyo, Keith E. J.; Liu, Zihe; Magnusson, Ylva

    2014-01-01

    Protein titers, a key bioprocessing metric, depend both on the synthesis of protein and the degradation of protein. Secreted recombinant protein production in Saccharomyces cerevisiae is an attractive platform as minimal media can be used for cultivation, thus reducing fermentation costs...... and transcriptomics, we identify metabolic and regulatory markers that are consistent with uptake of whole proteins by endocytosis, followed by intracellular degradation and catabolism of substituent amino acids. Uptake and degradation of recombinant protein products may be common in S. cerevisiae protein secretion...... and simplifying downstream purification, compared to other systems that require complex media. As such, engineering S. cerevisiae to improve titers has been then the subject of significant attention, but the majority of previous efforts have been focused on improving protein synthesis. Here, we characterize...

  10. The RTR complex as caretaker of genome stability and its unique meiotic function in plants

    Directory of Open Access Journals (Sweden)

    Alexander eKnoll

    2014-02-01

    Full Text Available The RTR complex consisting of a RecQ helicase, a type IA topoisomerase and the structural protein RMI1 is involved in the processing of DNA recombination intermediates in all eukaryotes. In Arabidopsis thaliana the complex partners RECQ4A, topoisomerase 3α and RMI1 have been shown to be involved in DNA repair and in the suppression of homologous recombination (HR in somatic cells. Interestingly, mutants of AtTOP3A and AtRMI1 are also sterile due to extensive chromosome breakage in meiosis I, a phenotype that seems to be specific for plants. Although both proteins are essential for meiotic recombination it is still elusive on what kind of intermediates they are acting on. Recent data indicate that the pattern of non-crossover (NCO-associated meiotic gene conversion (GC differs between plants and other eukaryotes, as less NCOs in comparison to crossovers (CO could be detected in Arabidopsis. This indicates that NCOs happen either more rarely in plants or that the conversion tract length is significantly shorter than in other organisms. As the TOP3α/RMI1-mediated dissolution of recombination intermediates results exclusively in NCOs, we suggest that the peculiar GC pattern found in plants is connected to the unique role, members of the RTR complex play in plant meiosis.

  11. High-level transient expression of recombinant protein in lettuce.

    Science.gov (United States)

    Joh, Lawrence D; Wroblewski, Tadeusz; Ewing, Nicholas N; VanderGheynst, Jean S

    2005-09-30

    Transient expression following agroinfiltration of plant tissue was investigated as a system for producing recombinant protein. As a model system, Agrobacterium tumefaciens containing the beta-glucuronidase (GUS) gene was vacuum infiltrated into lettuce leaf disks. Infiltration with a suspension of 10(9) colony forming units/mL followed by incubation for 72 h at 22 degrees C in continuous darkness produced a maximum of 0.16% GUS protein based on dry tissue or 1.1% GUS protein based on total soluble protein. This compares favorably to expression levels for commercially manufactured GUS protein from transgenic corn seeds. A. tumefaciens culture medium pH between 5.6 and 7.0 and surfactant concentrations lettuce to produce GUS protein more rapidly, but final levels did not exceed the GUS production in leaves incubated in continuous darkness after 72 h at 22 degrees C. The kinetics of GUS expression during incubation in continuous light and dark were represented well using a logistic model, with rate constants of 0.30 and 0.29/h, respectively. To semi-quantitatively measure the GUS expression in large numbers of leaf disks, a photometric enhancement of the standard histochemical staining method was developed. A linear relationship with an R2 value of 0.90 was determined between log10 (% leaf darkness) versus log10 (GUS activity). Although variability in expression level was observed, agroinfiltration appears to be a promising technology that could potentially be scaled up to produce high-value recombinant proteins in planta. Copyright 2005 Wiley Periodicals, Inc

  12. Dynamic organization of genetic recombination proteins and chromosomes

    International Nuclear Information System (INIS)

    Essers, J.; Van Cappellen, G.; Van Drunen, E.; Theil, A.; Jaspers, N.N.G.J.; Houtsmuller, A.B.; Vermeulen, W.; Kanaar, R.

    2003-01-01

    Homologous recombination requires the co-ordinated action of the RAD52 group proteins, including Rad51, Rad52 and Rad54. Upon treatment of mammalian cells with ionizing radiation, these proteins accumulate into foci at sites of DSB induction. We probed the nature of the DNA damage-induced foci in living cells with the use of photobleaching techniques. These foci are not static assemblies of DNA repair proteins. Instead, they are dynamic structures of which Rad51 is a stable core component, while Rad52 and Rad54 reversibly interact with the structure. Furthermore, even though the RAD52 group proteins colocalize in the DNA damage-induced foci, the majority of the proteins are not part of the same multi-protein complex in the absence of DNA damage. Executing DNA transactions through dynamic multi-protein complexes, rather than stable holo-complexes, allows greater flexibility during the transaction. In case of DNA repair, for example, it allows cross talk between different DNA repair pathways and coupling to other DNA transactions, such as replication. In addition to the behavior of proteins in living cells, we have tracked chromosomes during cell division. Our results suggest that the relative position of chromosomes in the mother cell is conserved in its daughter cells

  13. The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans.

    Science.gov (United States)

    Manhart, Carol M; Ni, Xiaodan; White, Martin A; Ortega, Joaquin; Surtees, Jennifer A; Alani, Eric

    2017-04-01

    Crossing over between homologs is initiated in meiotic prophase by the formation of DNA double-strand breaks that occur throughout the genome. In the major interference-responsive crossover pathway in baker's yeast, these breaks are resected to form 3' single-strand tails that participate in a homology search, ultimately forming double Holliday junctions (dHJs) that primarily include both homologs. These dHJs are resolved by endonuclease activity to form exclusively crossovers, which are critical for proper homolog segregation in Meiosis I. Recent genetic, biochemical, and molecular studies in yeast are consistent with the hypothesis of Mlh1-Mlh3 DNA mismatch repair complex acting as the major endonuclease activity that resolves dHJs into crossovers. However, the mechanism by which the Mlh1-Mlh3 endonuclease is activated is unknown. Here, we provide evidence that Mlh1-Mlh3 does not behave like a structure-specific endonuclease but forms polymers required to generate nicks in DNA. This conclusion is supported by DNA binding studies performed with different-sized substrates that contain or lack polymerization barriers and endonuclease assays performed with varying ratios of endonuclease-deficient and endonuclease-proficient Mlh1-Mlh3. In addition, Mlh1-Mlh3 can generate religatable double-strand breaks and form an active nucleoprotein complex that can nick DNA substrates in trans. Together these observations argue that Mlh1-Mlh3 may not act like a canonical, RuvC-like Holliday junction resolvase and support a novel model in which Mlh1-Mlh3 is loaded onto DNA to form an activated polymer that cleaves DNA.

  14. The roles of the Saccharomyces cerevisiae RecQ helicase SGS1 in meiotic genome surveillance.

    Directory of Open Access Journals (Sweden)

    Amit Dipak Amin

    2010-11-01

    Full Text Available The Saccharomyces cerevisiae RecQ helicase Sgs1 is essential for mitotic and meiotic genome stability. The stage at which Sgs1 acts during meiosis is subject to debate. Cytological experiments showed that a deletion of SGS1 leads to an increase in synapsis initiation complexes and axial associations leading to the proposal that it has an early role in unwinding surplus strand invasion events. Physical studies of recombination intermediates implicate it in the dissolution of double Holliday junctions between sister chromatids.In this work, we observed an increase in meiotic recombination between diverged sequences (homeologous recombination and an increase in unequal sister chromatid events when SGS1 is deleted. The first of these observations is most consistent with an early role of Sgs1 in unwinding inappropriate strand invasion events while the second is consistent with unwinding or dissolution of recombination intermediates in an Mlh1- and Top3-dependent manner. We also provide data that suggest that Sgs1 is involved in the rejection of 'second strand capture' when sequence divergence is present. Finally, we have identified a novel class of tetrads where non-sister spores (pairs of spores where each contains a centromere marker from a different parent are inviable. We propose a model for this unusual pattern of viability based on the inability of sgs1 mutants to untangle intertwined chromosomes. Our data suggest that this role of Sgs1 is not dependent on its interaction with Top3. We propose that in the absence of SGS1 chromosomes may sometimes remain entangled at the end of pre-meiotic replication. This, combined with reciprocal crossing over, could lead to physical destruction of the recombined and entangled chromosomes. We hypothesise that Sgs1, acting in concert with the topoisomerase Top2, resolves these structures.This work provides evidence that Sgs1 interacts with various partner proteins to maintain genome stability throughout

  15. Osteoinductive recombinant silk fusion proteins for bone regeneration.

    Science.gov (United States)

    Dinjaski, Nina; Plowright, Robyn; Zhou, Shun; Belton, David J; Perry, Carole C; Kaplan, David L

    2017-02-01

    Protein polymers provide a unique opportunity for tunable designs of material systems due to the genetic basis of sequence control. To address the challenge of biomineralization interfaces with protein based materials, we genetically engineered spider silks to design organic-inorganic hybrid systems. The spider silk inspired domain (SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQGT) 15 served as an organic scaffold to control material stability and to allow multiple modes of processing, whereas the hydroxyapatite binding domain VTKHLNQISQSY (VTK), provided control over osteogenesis. The VTK domain was fused either to the N-, C- or both terminals of the spider silk domain to understand the effect of position on material properties and mineralization. The addition of the VTK domain to silk did not affect the physical properties of the silk recombinant constructs, but it had a critical role in the induction of biomineralization. When the VTK domain was placed on both the C- and N-termini the formation of crystalline hydroxyapatite was significantly increased. In addition, all of the recombinant proteins in film format supported the growth and proliferation of human mesenchymal stem cells (hMSCs). Importantly, the presence of the VTK domain enhanced osteoinductive properties up to 3-fold compared to the control (silk alone without VTK). Therefore, silk-VTK fusion proteins have been shown suitable for mineralization and functionalization for specific biomedical applications. Organic-inorganic interfaces are integral to biomaterial functions in many areas of repair and regeneration. Several protein polymers have been investigated for this purpose. Despite their success the limited options to fine-tune their material properties, degradation patterns and functionalize them for each specific biomedical application limits their application. Various studies have shown that the biological performance of such proteins can be improved by genetic engineering. The present study provides data

  16. Recombinant proteins in therapeutics: haemophilia treatment as an example

    Directory of Open Access Journals (Sweden)

    Liras Antonio

    2008-04-01

    Full Text Available Abstract One of the most spectacular advances in the history of scientific knowledge was the discovery of deoxyribonucleic acid (DNA by Watson and Crick in 1953. This enabled certain proteins to be prepared in this way for their therapeutic use in clinical practice. Today, in the first decade of the 21st century, hundreds of therapeutic proteins have been produced recombinantly and about 50 of them have been approved for clinical use. Because of the specific procedure used for obtaining these products, which is based on expressing a atherapeutica gene from a fragment of DNA in a cell to produce a functional protein that is free from any human or animal component, they are especially acleana and thus the therapy of choice for many current diseases. The immediate question is: why are recombinant products not used more extensively given their high efficacy and maximum safety? In short, we are faced with an interesting but also unfortunate paradox of pharmacology that greater progress in therapeutic procedures is not always associated with greater introduction of those resources that are safest, for the simple reason that they are more costly.

  17. Utilizing protein-lean coproducts from corn containing recombinant pharmaceutical proteins for ethanol production.

    Science.gov (United States)

    Paraman, Ilankovan; Moeller, Lorena; Scott, M Paul; Wang, Kan; Glatz, Charles E; Johnson, Lawrence A

    2010-10-13

    Protein-lean fractions of corn (maize) containing recombinant (r) pharmaceutical proteins were evaluated as a potential feedstock to produce fuel ethanol. The levels of residual r-proteins in the coproduct, distillers dry grains with solubles (DDGS), were determined. Transgenic corn lines containing recombinant green fluorescence protein (r-GFP) and a recombinant subunit vaccine of Escherichia coli enterotoxin (r-LTB), primarily expressed in endosperm, and another two corn lines containing recombinant human collagen (r-CIα1) and r-GFP, primarily expressed in germ, were used as model systems. The kernels were either ground and used for fermentation or dry fractionated to recover germ-rich fractions prior to grinding for fermentation. The finished beers of whole ground kernels and r-protein-spent endosperm solids contained 127-139 and 138-155 g/L ethanol concentrations, respectively. The ethanol levels did not differ among transgenic and normal corn feedstocks, indicating the residual r-proteins did not negatively affect ethanol production. r-Protein extraction and germ removal also did not negatively affect fermentation of the remaining mass. Most r-proteins were inactivated during the mashing process used to prepare corn for fermentation. No functionally active r-GFP or r-LTB proteins were found after fermentation of the r-protein-spent solids; however, a small quantity of residual r-CIα1 was detected in DDGS, indicating that the safety of DDGS produced from transgenic grain for r-protein production needs to be evaluated for each event. Protease treatment during fermentation completely hydrolyzed the residual r-CIα1, and no residual r-proteins were detectable in DDGS.

  18. [Eukaryotic Expression and Immunogenic Research of Recombination Ebola Virus Membrane Protein Gp-Fc].

    Science.gov (United States)

    Zhang, Xiaoguang; Yang, Ren; Wang, Jiao; Wang, Xuan; Hou, Mieling; An, Lina; Zhu, Ying; Cao, Yuxi; Zeng, Yi

    2016-01-01

    We used 293 cells to express the recombinant membrane protein of the Ebola virus. Then, the immunogenicity of the recombinant protein was studied by immunized BALB/c mice. According to the codon use frequency of humans, the gene encoding the extracellular domain of the Ebola virus membrane protein was optimized, synthesized, and inserted into the eukaryotic expression plasmid pXG-Fc to construct the human IgG Fc and Ebola GP fusion protein expression plasmid pXG-modGP-Fc. To achieve expression, the fusion protein expression vector was transfected into high-density 293 cells using transient transfection technology. The recombinant protein was purified by protein A affinity chromatography. BALB/c mice were immunized with the purified fusion protein, and serum antibody titers evaluated by an indirect enzyme-linked immunosorbent assay (ELISA). Purification and analyses of the protein revealed that the eukaryotic expression vector could express the recombinant protein GP-Fc effectively, and that the recombinant protein in the supernatant of the cell culture was present as a dimer. After immunization with the purified recombinant protein, a high titer of antigen-specific IgG could be detected in the serum of immunized mice by indirect ELISA, showing that the recombinant protein had good immunogenicity. These data suggest that we obtained a recombinant protein with good immunogenicity. Our study is the basis for development of a vaccine against the Ebola virus and for screening of monoclonal antibodies.

  19. DMC1 functions in a Saccharomyces cerevisiae meiotic pathway that is largely independent of the RAD51 pathway

    International Nuclear Information System (INIS)

    Dresser, M.E.; Ewing, D.J.; Conrad, M.N.; Dominguez, A.M.; Barstead, R.; Jiang, H.; Kodadek, T.

    1997-01-01

    Meiotic recombination in the yeast Saccharomyces cerevisiae requires two similar recA-like proteins, Dmc1p and Rad51p. A screen for dominant meiotic mutants provided DMC1-G126D, a dominant allele mutated in the conserved ATP-binding site (specifically, the A-loop motif) that confers a null phenotype. A recessive null allele, dmc1-K69E, was isolated as an intragenic suppressor of DMC1-G126D. Dmc1-K69Ep, unlike Dmc1p, does not interact homotypically in a two-hybrid assay, although it does interact with other fusion proteins identified by two-hybrid screen with Dmc1p. Dmc1p, unlike Rad51p, does not interact in the two-hybrid assay with Rad52p or Rad54p. However, Dmc1p does interact with Tid1p, a Rad54p homologue, with Tid4p, a Rad16p homologue, and with other fusion proteins that do not interact with Rad51p, suggesting that Dmc1p and Rad51p function in separate, though possibly overlapping, recombinational repair complexes. Epistasis analysis suggests that DMC1 and RAD51 function in separate pathways responsible for meiotic recombination. Taken together, our results are consistent with a requirement for DMC1 for meiosis-specific entry of DNA double-strand break ends into chromatin. Interestingly, the pattern on CHEF gels of chromosome fragments that result from meiotic DNA double-strand break formation is different in DMC1 mutant strains from that seen in rad50S strains. (author)

  20. Recombinant human prion protein inhibits prion propagation in vitro.

    Science.gov (United States)

    Yuan, Jue; Zhan, Yi-An; Abskharon, Romany; Xiao, Xiangzhu; Martinez, Manuel Camacho; Zhou, Xiaochen; Kneale, Geoff; Mikol, Jacqueline; Lehmann, Sylvain; Surewicz, Witold K; Castilla, Joaquín; Steyaert, Jan; Zhang, Shulin; Kong, Qingzhong; Petersen, Robert B; Wohlkonig, Alexandre; Zou, Wen-Quan

    2013-10-09

    Prion diseases are associated with the conformational conversion of the cellular prion protein (PrP(C)) into the pathological scrapie isoform (PrP(Sc)) in the brain. Both the in vivo and in vitro conversion of PrP(C) into PrP(Sc) is significantly inhibited by differences in amino acid sequence between the two molecules. Using protein misfolding cyclic amplification (PMCA), we now report that the recombinant full-length human PrP (rHuPrP23-231) (that is unglycosylated and lacks the glycophosphatidylinositol anchor) is a strong inhibitor of human prion propagation. Furthermore, rHuPrP23-231 also inhibits mouse prion propagation in a scrapie-infected mouse cell line. Notably, it binds to PrP(Sc), but not PrP(C), suggesting that the inhibitory effect of recombinant PrP results from blocking the interaction of brain PrP(C) with PrP(Sc). Our findings suggest a new avenue for treating prion diseases, in which a patient's own unglycosylated and anchorless PrP is used to inhibit PrP(Sc) propagation without inducing immune response side effects.

  1. Molecular Basis for Enhancement of the Meiotic DMCI Recombinase by RAD51AP1

    Energy Technology Data Exchange (ETDEWEB)

    Dray, Eloise; Dunlop, Myun Hwa; Kauppi, Liisa; San Filippo, Joseph San; Wiese, Claudia; Tsai, Miaw-Sheue; Begovic, Sead; Schild, David; Jasin, Maria; Keeney, Scott; Sung, Patrick

    2010-11-05

    Homologous recombination is needed for meiotic chromosome segregation, genome maintenance, and tumor suppression. RAD51AP1 (RAD51 Associated Protein 1) has been shown to interact with and enhance the recombinase activity of RAD51. Accordingly, genetic ablation of RAD51AP1 leads to enhanced sensitivity to and also chromosome aberrations upon DNA damage, demonstrating a role for RAD51AP1 in mitotic homologous recombination. Here we show physical association of RAD51AP1 with the meiosis-specific recombinase DMC1 and a stimulatory effect of RAD51AP1 on the DMC1-mediated D-loop reaction. Mechanistic studies have revealed that RAD51AP1 enhances the ability of the DMC1 presynaptic filament to capture the duplex DNA partner and to assemble the synaptic complex, in which the recombining DNA strands are homologously aligned. We also provide evidence that functional co-operation is dependent on complex formation between DMC1 and RAD51AP1, and that distinct epitopes in RAD51AP1 mediate interactions with RAD51 and DMC1. Finally, we show that RAD51AP1 is expressed in mouse testes, and that RAD51AP1 foci co-localize with a subset of DMC1 foci in spermatocytes. These results suggest that RAD51AP1 also serves an important role in meiotic homologous recombination.

  2. Purification and Characterization of Recombinant Vaccinia L1R Protein from Escherichia coli

    Science.gov (United States)

    2016-08-01

    RECOMBINANT VACCINIA L1R PROTEIN FROM ESCHERICHIA COLI 1. INTRODUCTION 1.1 Background Vaccinia virus (VACV) is the active component of the...the preparation of the recombinant VACV L1R protein fragment by denaturing , refolding, and purifying material expressed into inclusion bodies in...PURIFICATION AND CHARACTERIZATION OF RECOMBINANT VACCINIA L1R PROTEIN FROM ESCHERICHIA COLI ECBC-TR-1370

  3. Cloning and expression of the recombinant NP24I protein from ...

    African Journals Online (AJOL)

    Jane

    2011-10-24

    Oct 24, 2011 ... protein from tomato fruit and study of its antimicrobial ... the recombinant NP24, as well as to prove the activity of native protein on the bacterial as well as fungal .... The antifungal effect of the recombinant NP24I protein was.

  4. Analysis of meiosis in SUN1 deficient mice reveals a distinct role of SUN2 in mammalian meiotic LINC complex formation and function.

    Directory of Open Access Journals (Sweden)

    Jana Link

    2014-02-01

    Full Text Available LINC complexes are evolutionarily conserved nuclear envelope bridges, composed of SUN (Sad-1/UNC-84 and KASH (Klarsicht/ANC-1/Syne/homology domain proteins. They are crucial for nuclear positioning and nuclear shape determination, and also mediate nuclear envelope (NE attachment of meiotic telomeres, essential for driving homolog synapsis and recombination. In mice, SUN1 and SUN2 are the only SUN domain proteins expressed during meiosis, sharing their localization with meiosis-specific KASH5. Recent studies have shown that loss of SUN1 severely interferes with meiotic processes. Absence of SUN1 provokes defective telomere attachment and causes infertility. Here, we report that meiotic telomere attachment is not entirely lost in mice deficient for SUN1, but numerous telomeres are still attached to the NE through SUN2/KASH5-LINC complexes. In Sun1(-/- meiocytes attached telomeres retained the capacity to form bouquet-like clusters. Furthermore, we could detect significant numbers of late meiotic recombination events in Sun1(-/- mice. Together, this indicates that even in the absence of SUN1 telomere attachment and their movement within the nuclear envelope per se can be functional.

  5. A Generic Protocol for Intracellular Expression of Recombinant Proteins in Bacillus subtilis.

    Science.gov (United States)

    Phan, Trang; Huynh, Phuong; Truong, Tuom; Nguyen, Hoang

    2017-01-01

    Bacillus subtilis (B. subtilis) is a potential and attractive host for the production of recombinant proteins. Different expression systems for B. subtilis have been developed recently, and various target proteins have been recombinantly synthesized and purified using this host. In this chapter, we introduce a generic protocol to express a recombinant protein in B. subtilis. It includes protocols for (1) using our typical expression vector (plasmid pHT254) to introduce a target gene, (2) transformation of the target vector into B. subtilis, and (3) evaluation of the actual expression of a recombinant protein.

  6. Recombiner

    International Nuclear Information System (INIS)

    Kikuchi, Nobuo.

    1983-01-01

    Purpose: To shorten the pre-heating time for a recombiner and obtain a uniform temperature distribution for the charged catalyst layer in a BWR type reactor. Constitution: A pre-heating heater is disposed to the outer periphery of a vessel for a recombiner packed with catalysts for recombining hydrogen and oxygen in gases flowing through a radioactive gaseous wastes processing system. Heat pipes for transmitting the heat applied to said container to the catalyst are disposed vertically and horizontally within the container. Different length of the heat pipes are combined. In this way, pre-heating time for the recombiner before the operation start and before the system switching can be shortened and the uniform pre-heating for the inside of the recombiner is also made possible. Further, heater control in the pre-heating can be carried out effectively and with ease. (Moriyama, K.)

  7. Prdm9 controls activation of mammalian recombination hotspots.

    Science.gov (United States)

    Parvanov, Emil D; Petkov, Petko M; Paigen, Kenneth

    2010-02-12

    Mammalian meiotic recombination, which preferentially occurs at specialized sites called hotspots, ensures the orderly segregation of meiotic chromosomes and creates genetic variation among offspring. A locus on mouse chromosome 17, which controls activation of recombination at multiple distant hotspots, has been mapped within a 181-kilobase interval, three of whose genes can be eliminated as candidates. The remaining gene, Prdm9, codes for a zinc finger containing histone H3K4 trimethylase that is expressed in early meiosis and whose deficiency results in sterility in both sexes. Mus musculus exhibits five alleles of Prdm9; human populations exhibit two predominant alleles and multiple minor alleles. The identification of Prdm9 as a protein regulating mammalian recombination hotspots initiates molecular studies of this important biological control system.

  8. Recombiner

    International Nuclear Information System (INIS)

    Osumi, Morimichi.

    1979-01-01

    Purpose: To provide a recombiner which is capable of converting hydrogen gas into water by use of high-frequency heating at comparatively low temperatures and is safe and cheap in cost. Constitution: Hydrogen gas is introduced from an outer pipeline to the main structure of a recombiner, and when it passes through the vicinity of the central part of the recombiner, it is reacted with copper oxide (CuO 2 ) heated to a temperature more than 300 0 C by a high-frequency heater, and converted gently into water by reduction operation (2H 2 + CuO 2 → Cu + 2H 2 O). The thus prepared water is exhausted through the outer pipeline to a suppression pool. A part of hydrogen gas which has not been converted completely into water by the reaction and is remaining as hydrogen is recovered through exhaust nozzles and again introduced into the main structure of the recombiner. (Yoshino, Y.)

  9. Rec8p, a meiotic recombination and sister chromatid cohesion phosphoprotein of the Rad21p family conserved from fision yeast to humans.

    NARCIS (Netherlands)

    S. Parisi; M.J. McKay (Michael); M. Molnar; M.A. Thompson (Anne); P.J. van der Spek (Peter); E. van Drunen-Schoenmaker; R. Kanaar (Roland); E. Lehmann; J.H.J. Hoeijmakers (Jan); J. Kohli

    1999-01-01

    textabstractOur work and that of others defined mitosis-specific (Rad21 subfamily) and meiosis-specific (Rec8 subfamily) proteins involved in sister chromatid cohesion in several eukaryotes, including humans. Mutation of the fission yeast Schizosaccharomyces pombe rec8 gene was previously shown to

  10. Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells.

    Science.gov (United States)

    Fabrick, Jeffrey A; Hull, J Joe

    2017-04-20

    Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

  11. Recombinant proteins from plants: production and isolation of clinically useful compounds

    National Research Council Canada - National Science Library

    Cunningham, Charles; Porter, Andrew J. R

    1998-01-01

    ... of recombinant proteins for use as specialist industrial or therapeutic biomolecules. The intention of Recombinant Proteins from Plants is to provide comprehensive and detailed protocols covering all the latest molecular approaches. Because the production oftransgenic plants has become routine in many laboratories, coverage is also given to some of the more "...

  12. Analysis of recombinant proteins by isoelectric focusing in immobilized pH gradients

    NARCIS (Netherlands)

    Bischoff, Rainer; Roecklin, D.; Roitsch, C.

    1992-01-01

    Isoelectric focusing in immobilized pH gradients (IEF-IPG) was used to analyze three different recombinant proteins. Recombinant leech hirudin (65 amino acids, three disulfide bonds) expressed in Saccharomyces cerevisiae as a secreted protein and purified by anion-exchange and reversed-phase

  13. Limiting factors in Escherichia colifed-batch production of recombinant proteins

    DEFF Research Database (Denmark)

    Sanden, A.M.; Prytz, I.; Tubelekas, I.

    2003-01-01

    recombinant protein production, fed-batch, specific growth rate, feed profile, induction, mRNA, transcription, translation, acetic acid formation......recombinant protein production, fed-batch, specific growth rate, feed profile, induction, mRNA, transcription, translation, acetic acid formation...

  14. Immunogenicity of recombinant feline infectious peritonitis virus spike protein in mice and kittens

    NARCIS (Netherlands)

    Horzinek, M.C.; Vennema, H.; Groot, R. de; Harbour, D.A.; Dalderup, M.; Gruffydd-Jones, T.; Spaan, W.J.M.

    1990-01-01

    The gene encoding the fusogenic spike protein of the coronavirus causing feline infectious peritonitis (FIVP) was recombined into the genome of vaccinia virus, strain WR. The recombinant induced spike protein specific, in vitro neutralizing antibodies in mkice. When kittens were immunized with the

  15. Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.

    Science.gov (United States)

    Morris, Jacqueline; Jayanthi, Srinivas; Langston, Rebekah; Daily, Anna; Kight, Alicia; McNabb, David S; Henry, Ralph; Kumar, Thallapuranam Krishnaswamy Suresh

    2016-10-01

    Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a

  16. Mutations in the RNA-binding domains of tombusvirus replicase proteins affect RNA recombination in vivo

    International Nuclear Information System (INIS)

    Panaviene, Zivile; Nagy, Peter D.

    2003-01-01

    RNA recombination, which is thought to occur due to replicase errors during viral replication, is one of the major driving forces of virus evolution. In this article, we show evidence that the replicase proteins of Cucumber necrosis virus, a tombusvirus, are directly involved in RNA recombination in vivo. Mutations within the RNA-binding domains of the replicase proteins affected the frequency of recombination observed with a prototypical defective-interfering (DI) RNA, a model template for recombination studies. Five of the 17 replicase mutants tested showed delay in the formation of recombinants when compared to the wild-type helper virus. Interestingly, two replicase mutants accelerated recombinant formation and, in addition, these mutants also increased the level of subgenomic RNA synthesis (Virology 308 (2003), 191-205). A trans-complementation system was used to demonstrate that mutation in the p33 replicase protein resulted in altered recombination rate. Isolated recombinants were mostly imprecise (nonhomologous), with the recombination sites clustered around a replication enhancer region and a putative cis-acting element, respectively. These RNA elements might facilitate the proposed template switching events by the tombusvirus replicase. Together with data in the article cited above, results presented here firmly establish that the conserved RNA-binding motif of the replicase proteins is involved in RNA replication, subgenomic RNA synthesis, and RNA recombination

  17. High-yield secretion of recombinant proteins from the microalga Chlamydomonas reinhardtii

    DEFF Research Database (Denmark)

    Ramos Martinez, Erick Miguel; Fimognari, Lorenzo; Sakuragi, Yumiko

    2017-01-01

    Microalga-based biomanufacturing of recombinant proteins is attracting growing attention due to its advantages in safety, metabolic diversity, scalability and sustainability. Secretion of recombinant proteins can accelerate the use of microalgal platforms by allowing post......-translational modifications and easy recovery of products from the culture media. However, currently, the yields of secreted recombinant proteins are low, which hampers the commercial application of this strategy. This study aimed at expanding the genetic tools for enhancing secretion of recombinant proteins in Chlamydomonas...... in the endoplasmic reticulum (ER). Taken together, the results demonstrate the utility of the gametolysin signal sequence and (SP)n glycomodule to promote a more efficient biomanufacturing of microalgae-based recombinant proteins....

  18. Cloning an artificial gene encoding angiostatic anginex: From designed peptide to functional recombinant protein

    International Nuclear Information System (INIS)

    Brandwijk, Ricardo J.M.G.E.; Nesmelova, Irina; Dings, Ruud P.M.; Mayo, Kevin H.; Thijssen, Victor L.J.L.; Griffioen, Arjan W.

    2005-01-01

    Anginex, a designed peptide 33-mer, is a potent angiogenesis inhibitor and anti-tumor agent in vivo. Anginex functions by inhibiting endothelial cell (EC) proliferation and migration leading to detachment and apoptosis of activated EC's. To better understand tumor endothelium targeting properties of anginex and enable its use in gene therapy, we constructed an artificial gene encoding the biologically exogenous peptide and produced the protein recombinantly in Pichia pastoris. Mass spectrometry shows recombinant anginex to be a dimer and circular dichroism shows the recombinant protein folds with β-strand structure like the synthetic peptide. Moreover, like parent anginex, the recombinant protein is active at inhibiting EC growth and migration, as well as inhibiting angiogenesis in vivo in the chorioallantoic membrane of the chick embryo. This study demonstrated that it is possible to produce a functionally active protein version of a rationally designed peptide, using an artificial gene and the recombinant protein approach

  19. Selective Advantage of Recombination in Evolving Protein Populations:. a Lattice Model Study

    Science.gov (United States)

    Williams, Paul D.; Pollock, David D.; Goldstein, Richard A.

    Recent research has attempted to clarify the contributions of several mutational processes, such as substitutions or homologous recombination. Simplistic, tractable protein models, which determine the compact native structure phenotype from the sequence genotype, are well-suited to such studies. In this paper, we use a lattice-protein model to examine the effects of point mutation and homologous recombination on evolving populations of proteins. We find that while the majority of mutation and recombination events are neutral or deleterious, recombination is far more likely to be beneficial. This results in a faster increase in fitness during evolution, although the final fitness level is not significantly changed. This transient advantage provides an evolutionary advantage to subpopulations that undergo recombination, allowing fixation of recombination to occur in the population.

  20. The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection.

    Directory of Open Access Journals (Sweden)

    Qingwen Jin

    Full Text Available Insertion of T4 lysozyme (T4L into the GPCR successfully enhanced GPCR protein stability and solubilization. However, the biological functions of the recombinant GPCR protein have not been analyzed.We engineered the CCR5-T4L mutant and expressed and purified the soluble recombinant protein using an E.coli expression system. The antiviral effects of this recombinant protein in THP-1 cell lines, primary human macrophages, and PBMCs from different donors were investigated. We also explored the possible mechanisms underlying the observed antiviral effects.Our data showed the biphasic inhibitory and promotion effects of different concentrations of soluble recombinant CCR5-T4L protein on R5 tropic human immunodeficiency virus-1 (HIV-1 infection in THP-1 cell lines, human macrophages, and PBMCs from clinical isolates. We demonstrated that soluble recombinant CCR5-T4L acts as a HIV-1 co-receptor, interacts with wild type CCR5, down-regulates the surface CCR5 expression in human macrophages, and interacts with CCL5 to inhibit macrophage migration. Using binding assays, we further determined that recombinant CCR5-T4L and [125I]-CCL5 compete for the same binding site on wild type CCR5.Our results suggest that recombinant CCR5-T4L protein marginally promotes HIV-1 infection at low concentrations and markedly inhibits infection at higher concentrations. This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents.

  1. Meiotic faults as a major cause of offspring inviability

    DEFF Research Database (Denmark)

    Levitis, Daniel; Zimmerman, Kolea; Pringle, Anne

    2014-01-01

    , this result demonstrates that failures associated with meiosis are a major cause of offspring inviability not only for meiotic parthenogenesis, but for sexual reproducers such as humans. Meiosis is necessary for genetic recombination in eukaryotes, but is vestigial, and costly, in parthenogens. The question...... range of organisms....

  2. Serological diagnosis of Strongylus vulgaris infection: use of a recombinant protein

    DEFF Research Database (Denmark)

    Andersen, Ulla Vestergaard; Howe, Daniel K.; Olsen, Susanne Nautrup

    , an immunoreactive cDNA clone was subcloned into E. coli and the plasmid sequenced, the open reading frame encoding the mature protein was cloned into a pET22b expression vector and expressed as a His-tagged recombinant protein in BL21 expression cells. The recombinant protein was used in an indirect enzyme....... vulgaris (n=9) reacted against the recombinant protein, expressed as optic density (OD) readings of >24 % of a positive control, while sera from negative horses had OD readings

  3. The fate and role of macromolecules synthesized during mammalian oocyte meiotic maturation. I. Autoradiographic topography of newly synthesized RNA and protein in the germinal vesicle of the pig and rabbit

    International Nuclear Information System (INIS)

    Motlik, J.; Kopecny, V.; Pivko, J.

    1978-01-01

    Pig and rabbit oocytes were cytoautoradiographically checked for their synthetic activities during meiotic maturation. Tritiated uridine and lysine or 35 S-methionine were introduced into the culture medium in which the oocytes were maintained either immediately at the beginning of the germinal vesicle breakdown in vitro or after reaching a more advanced stage of this process in vitro or in vivo. Some oocytes were maintained thereafter in a cold medium to trace the metabolism of the labelled protein. In addition to uridine- 3 H incorporation into the nucleolus and nucleoplasm, during pig oocyte maturation it was found that an intensive RNA synthesis site appeared in association with condensing chromocentres of the GV II. A considerable proportion of oocytes from slaughterhouse material did not show intensive GV activity in RNA synthesis during maturation in vitro. In the pig and rabbit oocyte it was shown that the newly synthesized 3 H-lysine-labelled protein accumulated to a high degree in the GV and in the nucleolus. The labelled protein accumulated in the GV up to the stage of GV IV (pig) and persisted during the chase period in the ooplasm; it was found to be associated with chromosomes of metaphase I (pig) or metaphase II (rabbit) of the meiotic division. The process of protein accumulation in the GV was not influenced by meiotic arrest during oocyte culture in autologous follicular fluid. A similar accumulation of the label in the GV was detected in oocytes which were cultured in a medium enriched by 35 S-methionine. In some oocytes the labelled protein failed to accumulate in the nucleolar area during maturation in vitro

  4. Application of native signal sequences for recombinant proteins secretion in Pichia pastoris

    DEFF Research Database (Denmark)

    Borodina, Irina; Do, Duy Duc; Eriksen, Jens C.

    Background Methylotrophic yeast Pichia pastoris is widely used for recombinant protein production, largely due to its ability to secrete correctly folded heterologous proteins to the fermentation medium. Secretion is usually achieved by cloning the recombinant gene after a leader sequence, where...... alpha‐mating factor (MF) prepropeptide from Saccharomyces cerevisiae is most commonly used. Our aim was to test whether signal peptides from P. pastoris native secreted proteins could be used to direct secretion of recombinant proteins. Results Eleven native signal peptides from P. pastoris were tested...... by optimization of expression of three different proteins in P. pastoris. Conclusions Native signal peptides from P. pastoris can be used to direct secretion of recombinant proteins. A novel USER‐based P. pastoris system allows easy cloning of protein‐coding gene with the promoter and leader sequence of choice....

  5. Recombiner

    International Nuclear Information System (INIS)

    Saalfrank, H.

    1985-01-01

    Air containing hydrogen can be oxidized by heating in a container called a recombiner, in order to avoid the collection of hydrogen. The container is long and a large number of straight heating bars are arranged in parallel in it and they are flanged to a lid. The heating bars are surrounded by tubes, in order to obtain good heat transfer by a narrow annular gap. (orig.) [de

  6. Production of Recombinant and Tagged Proteins in the Hyperthermophilic Archaeon Sulfolobus solfataricus

    NARCIS (Netherlands)

    Albers, S.-V.; Jonuscheit, M.; Dinkelaker, S.; Urich, T.; Kletzin, A.; Tampé, R.; Driessen, A.J.M.; Schleper, C.

    Many systems are available for the production of recombinant proteins in bacterial and eukaryotic model organisms, which allow us to study proteins in their native hosts and to identify protein-protein interaction partners. In contrast, only a few transformation systems have been developed for

  7. Protein expression of Myt272-3 recombinant clone and in silico ...

    African Journals Online (AJOL)

    Purpose: To investigate the expression of Myt272-3 recombinant protein and also to predict a possible protein vaccine candidate against Mycobacterium tuberculosis. Methods: Myt272-3 protein was expressed in pET30a+-Myt272-3 clone. The purity of the protein was determined using Dynabeads® His-Tag Isolation ...

  8. Recombination homeostasis of meiosis during spermatogenesis under nicotine treatment

    Directory of Open Access Journals (Sweden)

    Zhai Jingli

    2018-01-01

    Full Text Available Cigarette smoking can affect male fertility via the quality of semen. To explore the effects of nicotine, a major component of cigarettes, on meiotic recombination during spermatogenesis, C57BL/6J male mice were injected with nicotine at a dosage of 0.2 mg/100 g body weight daily for 35 days (nicotine-treated group; mice in the control group were injected with isopycnic normal saline. According to previous expression profiles of mouse sperm, a subset of meiosis-related genes was pooled using bioinformatic analysis. Protein expression was compared between the two groups using by Western blotting and immunohistochemistry. Recombination frequency during the meiosis phase of spermatogenesis was estimated by combined use of chromosome spread and immunofluorescence staining in mouse testes. Data mining analysis indicated that 4 genes that express meiotic topoisomerase-like protein SPO11, MutS protein homolog 4 (MSH4, strand exchange protein RAD51 and MutL protein homologue 1 (MLH1, were associated with the meiosis recombination process. The results of Western blotting and immunohistochemistry further showed that the protein expression of SPO11 (0.73-fold and MSH4 (0.73-fold was downregulated in murine testes after nicotine treatment, whereas the protein expression of both RAD51 (2.06-fold and MLH1 (1.40-fold was upregulated. Unexpectedly, we did not detect a significant difference in recombination frequency in meiosis during spermatogenesis in the nicotine-treated group as compared to the control. Taken together, these results indicate that nicotine can affect the expression profile of restructuring-related genes, but it does not significantly change the recombination frequency during male meiosis. These findings suggest there is a self-regulating mechanism during meiotic chromosome restructuring in male mice that responds to environmental stress.

  9. Companion Protease Inhibitors for the In Situ Protection of Recombinant Proteins in Plants.

    Science.gov (United States)

    Robert, Stéphanie; Jutras, Philippe V; Khalf, Moustafa; D'Aoust, Marc-André; Goulet, Marie-Claire; Sainsbury, Frank; Michaud, Dominique

    2016-01-01

    We previously described a procedure for the use of plant protease inhibitors as "companion" accessory proteins to prevent unwanted proteolysis of clinically useful recombinant proteins in leaf crude protein extracts (Benchabane et al. Methods Mol Biol 483:265-273, 2009). Here we describe the use of these inhibitors for the protection of recombinant proteins in planta, before their extraction from leaf tissues. A procedure is first described involving inhibitors co-expressed along-and co-migrating-with the protein of interest in host plant cells. An alternative, single transgene scheme is then described involving translational fusions of the recombinant protein and companion inhibitor. These approaches may allow for a significant improvement of protein steady-state levels in leaves, comparable to yield improvements observed with protease-deficient strains of less complex protein expression hosts such as E. coli or yeasts.

  10. Generation of monoclonal antibodies for the assessment of protein purification by recombinant ribosomal coupling

    DEFF Research Database (Denmark)

    Kristensen, Janni; Sperling-Petersen, Hans Uffe; Mortensen, Kim Kusk

    2005-01-01

    We recently described a conceptually novel method for the purification of recombinant proteins with a propensity to form inclusion bodies in the cytoplasm of Escherichia coli. Recombinant proteins were covalently coupled to the E. coli ribosome by fusing them to ribosomal protein 23 (rpL23...... therefore purified rpL23-GFP-His, rpL23-His and GFP from E. coli recombinants using affinity, ion exchange and hydrophobic interaction chromatography. These proteins could be purified with yields of 150, 150 and 1500 microg per gram cellular wet weight, respectively. However, rpL23-GFP-His could only...... proteolytic cleavage sites. We conclude that the generated antibodies can be used to evaluate ribosomal coupling of recombinant target proteins as well as the efficiency of their separation from the ribosome....

  11. A new potential secretion pathway for recombinant proteins in Bacillus subtilis.

    Science.gov (United States)

    Wang, Guangqiang; Xia, Yongjun; Gu, Zhennan; Zhang, Hao; Chen, Yong Q; Chen, Haiqin; Ai, Lianzhong; Chen, Wei

    2015-11-10

    Secretion of cytoplasmic expressed proteins into growth media has significant advantages. Due to the lack of an outer membrane, Bacillus subtilis is considered as a desirable 'cell factory' for the secretion of recombinant proteins. However, bottlenecks in the classical pathway for the secretion of recombinant proteins limit its use on a wide scale. In this study, we attempted to use four typical non-classically secreted proteins as signals to export three recombinant model proteins to the culture medium. All four non-classically secreted proteins can direct the export of the intrinsically disordered nucleoskeletal-like protein (Nsp). Two of them can guide the secretion of alkaline phosphatase (PhoA). One can lead the secretion of the thermostable β-galactosidase BgaB, which cannot be secreted with the aid of typical Sec-dependent signal peptides. Our results show that the non-classically secreted proteins lead the recombinant proteins to the culture medium, and thus non-classical protein secretion pathways can be exploited as a novel secretion pathway for recombinant proteins.

  12. New insights into the evolutionary origins of the recombination-activating gene proteins and V(D)J recombination.

    Science.gov (United States)

    Carmona, Lina Marcela; Schatz, David G

    2017-06-01

    The adaptive immune system of jawed vertebrates relies on V(D)J recombination as one of the main processes to generate the diverse array of receptors necessary for the recognition of a wide range of pathogens. The DNA cleavage reaction necessary for the assembly of the antigen receptor genes from an array of potential gene segments is mediated by the recombination-activating gene proteins RAG1 and RAG2. The RAG proteins have been proposed to originate from a transposable element (TE) as they share mechanistic and structural similarities with several families of transposases and are themselves capable of mediating transposition. A number of RAG-like proteins and TEs with sequence similarity to RAG1 and RAG2 have been identified, but only recently has their function begun to be characterized, revealing mechanistic links to the vertebrate RAGs. Of particular significance is the discovery of ProtoRAG, a transposon superfamily found in the genome of the basal chordate amphioxus. ProtoRAG has many of the sequence and mechanistic features predicted for the ancestral RAG transposon and is likely to be an evolutionary relative of RAG1 and RAG2. In addition, early observations suggesting that RAG1 is able to mediate V(D)J recombination in the absence of RAG2 have been confirmed, implying independent evolutionary origins for the two RAG genes. Here, recent progress in identifying and characterizing RAG-like proteins and the TEs that encode them is summarized and a refined model for the evolution of V(D)J recombination and the RAG proteins is presented. © 2016 Federation of European Biochemical Societies.

  13. Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF.

    Science.gov (United States)

    Cantu-Bustos, J Enrique; Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Galbraith, David W; McEvoy, Megan M; Zarate, Xristo

    2016-05-01

    Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Stabilizing additives added during cell lysis aid in the solubilization of recombinant proteins.

    Directory of Open Access Journals (Sweden)

    David J Leibly

    Full Text Available Insoluble recombinant proteins are a major issue for both structural genomics and enzymology research. Greater than 30% of recombinant proteins expressed in Escherichia coli (E. coli appear to be insoluble. The prevailing view is that insolubly expressed proteins cannot be easily solubilized, and are usually sequestered into inclusion bodies. However, we hypothesize that small molecules added during the cell lysis stage can yield soluble protein from insoluble protein previously screened without additives or ligands. We present a novel screening method that utilized 144 additive conditions to increase the solubility of recombinant proteins expressed in E. coli. These selected additives are natural ligands, detergents, salts, buffers, and chemicals that have been shown to increase the stability of proteins in vivo. We present the methods used for this additive solubility screen and detailed results for 41 potential drug target recombinant proteins from infectious organisms. Increased solubility was observed for 80% of the recombinant proteins during the primary and secondary screening of lysis with the additives; that is 33 of 41 target proteins had increased solubility compared with no additive controls. Eleven additives (trehalose, glycine betaine, mannitol, L-Arginine, potassium citrate, CuCl(2, proline, xylitol, NDSB 201, CTAB and K(2PO(4 solubilized more than one of the 41 proteins; these additives can be easily screened to increase protein solubility. Large-scale purifications were attempted for 15 of the proteins using the additives identified and eight (40% were prepared for crystallization trials during the first purification attempt. Thus, this protocol allowed us to recover about a third of seemingly insoluble proteins for crystallography and structure determination. If recombinant proteins are required in smaller quantities or less purity, the final success rate may be even higher.

  15. Escherichia coli fusion carrier proteins act as solubilizing agents for recombinant uncoupling protein 1 through interactions with GroEL

    International Nuclear Information System (INIS)

    Douette, Pierre; Navet, Rachel; Gerkens, Pascal; Galleni, Moreno; Levy, Daniel; Sluse, Francis E.

    2005-01-01

    Fusing recombinant proteins to highly soluble partners is frequently used to prevent aggregation of recombinant proteins in Escherichia coli. Moreover, co-overexpression of prokaryotic chaperones can increase the amount of properly folded recombinant proteins. To understand the solubility enhancement of fusion proteins, we designed two recombinant proteins composed of uncoupling protein 1 (UCP1), a mitochondrial membrane protein, in fusion with MBP or NusA. We were able to express soluble forms of MBP-UCP1 and NusA-UCP1 despite the high hydrophobicity of UCP1. Furthermore, the yield of soluble fusion proteins depended on co-overexpression of GroEL that catalyzes folding of polypeptides. MBP-UCP1 was expressed in the form of a non-covalent complex with GroEL. MBP-UCP1/GroEL was purified and characterized by dynamic light scattering, gel filtration, and electron microscopy. Our findings suggest that MBP and NusA act as solubilizing agents by forcing the recombinant protein to pass through the bacterial chaperone pathway in the context of fusion protein

  16. Recominant Pinoresino-Lariciresinol Reductase, Recombinant Dirigent Protein And Methods Of Use

    Science.gov (United States)

    Lewis, Norman G.; Davin, Laurence B.; Dinkova-Kostova, Albena T.; Fujita, Masayuki , Gang; David R. , Sarkanen; Simo , Ford; Joshua D.

    2003-10-21

    Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided from source species Forsythia intermedia, Thuja plicata, Tsuga heterophylla, Eucommia ulmoides, Linum usitatissimum, and Schisandra chinensis, which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.

  17. Recombinant Protein Production and Insect Cell Culture and Process

    Science.gov (United States)

    Spaulding, Glenn F. (Inventor); Goodwin, Thomas J. (Inventor); OConnor, Kim C. (Inventor); Francis, Karen M. (Inventor); Andrews, Angela D. (Inventor); Prewett, Tracey L. (Inventor)

    1997-01-01

    A process has been developed for recombinant production of selected polypeptides using transformed insect cells cultured in a horizontally rotating culture vessel modulated to create low shear conditions. A metabolically transformed insect cell line is produced using the culture procedure regardless of genetic transformation. The recombinant polypeptide can be produced by an alternative process using virtually infected or stably transformed insect cells containing a gene encoding the described polypeptide. The insect cells can also be a host for viral production.

  18. Assignment of the human gene for pregnancy-associated plasma protein A (PAPPA) to 9q33.1 by fluorescence in situ hybridization to mitotic and meiotic chromosomes

    DEFF Research Database (Denmark)

    Silahtaroglu, A N; Tümer, Z; Kristensen, Torsten

    1993-01-01

    Low levels of pregnancy-associated plasma protein A (PAPPA) during the first trimester has been suggested as a biochemical indicator of pregnancies with aneuploid fetuses. Furthermore, the complete absence of PAPPA in pregnancies associated with Cornelia de Lange syndrome (CL) has suggested...... a causal connection between PAPPA and the development of CL. We have assigned the locus for PAPPA to chromosome region 9q33.1 on mitotic and meiotic chromosomes by fluorescence in situ hybridization, using a 3.7-kb partial PAPPA cDNA probe...

  19. Refolding techniques for recovering biologically active recombinant proteins from inclusion bodies.

    Science.gov (United States)

    Yamaguchi, Hiroshi; Miyazaki, Masaya

    2014-02-20

    Biologically active proteins are useful for studying the biological functions of genes and for the development of therapeutic drugs and biomaterials in a biotechnology industry. Overexpression of recombinant proteins in bacteria, such as Escherichia coli, often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. As inclusion bodies contain relatively pure and intact proteins, protein refolding is an important process to obtain active recombinant proteins from inclusion bodies. However, conventional refolding methods, such as dialysis and dilution, are time consuming and, often, recovered yields of active proteins are low, and a trial-and-error process is required to achieve success. Recently, several approaches have been reported to refold these aggregated proteins into an active form. The strategies largely aim at reducing protein aggregation during the refolding procedure. This review focuses on protein refolding techniques using chemical additives and laminar flow in microfluidic chips for the efficient recovery of active proteins from inclusion bodies.

  20. Meiotic transmission of Drosophila pseudoobscura chromosomal arrangements.

    Directory of Open Access Journals (Sweden)

    Richard P Meisel

    Full Text Available Drosophila pseudoobscura harbors a rich gene arrangement polymorphism on the third chromosome generated by a series of overlapping paracentric inversions. The arrangements suppress recombination in heterokaryotypic individuals, which allows for the selective maintenance of coadapted gene complexes. Previous mapping experiments used to determine the degree to which recombination is suppressed in gene arrangement heterozygotes produced non-recombinant progeny in non-Mendelian ratios. The deviations from Mendelian expectations could be the result of viability differences between wild and mutant chromosomes, meiotic drive because of achiasmate pairing of homologues in heterokaryotypic females during meiosis, or a combination of both mechanisms. The possibility that the frequencies of the chromosomal arrangements in natural populations are affected by mechanisms other than adaptive selection led us to consider these hypotheses. We performed reciprocal crosses involving both heterozygous males and females to determine if the frequency of the non-recombinant progeny deviates significantly from Mendelian expectations and if the frequencies deviate between reciprocal crosses. We failed to observe non-Mendelian ratios in multiple crosses, and the frequency of the non-recombinant classes differed in only one of five pairs of reciprocal crosses despite sufficient power to detect these differences in all crosses. Our results indicate that deviations from Mendelian expectations in recombination experiments involving the D. pseudoobscura inversion system are most likely due to fitness differences of gene arrangement karyotypes in different environments.

  1. [Study on the anti-NTHi infection of Hap recombinant protein in vivo].

    Science.gov (United States)

    Li, Wan-yi; Wang, Bao-ning; Zuo, Feng-qiong; Zeng, Wei; Feng, Feng; Kuang, Yu; Jiang, Zhong-hua; Li, Ming-yuan

    2010-07-01

    To observe the immune effect of Hap recombinant protein on murine model of bronchopneumonia infected with NTHi, and explore the mechanism about the anti-NTHi infection. The C57BL/6 mice intranasally immunized with purified Hap recombinant protein and CT-B were challenged by NTHi encased in agar beads. The immunifaction of anti-infection was observed through encocyte counting of BALF, bacteria detection of lung and the pathologyical change of lung tissue. In the challenge with NTHi experiment, the inflammatory exudation of the infected murine and pathological change of lung tissue was relieved by combined immunization of Hap recombinant protein and CT-B, and quantity of NTHi in lung of the infected murine was reduced obviously. The Hap recombinant protein also had good ability of anti-NTHi infection in the murine model of NTHi bronchopneumonia. This study could offer the oretical and experimental basis for development of new vaccine against NTHi.

  2. Systems and methods for the secretion of recombinant proteins in gram negative bacteria

    Science.gov (United States)

    Withers, III, Sydnor T.; Dominguez, Miguel A; DeLisa, Matthew P.; Haitjema, Charles H.

    2016-08-09

    Disclosed herein are systems and methods for producing recombinant proteins utilizing mutant E. coli strains containing expression vectors carrying nucleic acids encoding the proteins, and secretory signal sequences to direct the secretion of the proteins to the culture medium. Host cells transformed with the expression vectors are also provided.

  3. Analysis of the ligand binding properties of recombinant bovine liver-type fatty acid binding protein

    DEFF Research Database (Denmark)

    Rolf, B; Oudenampsen-Krüger, E; Börchers, T

    1995-01-01

    The coding part of the cDNA for bovine liver-type fatty acid binding protein (L-FABP) has been amplified by RT-PCR, cloned and used for the construction of an Escherichia coli (E. coli) expression system. The recombinant protein made up to 25% of the soluble E. coli proteins and could be isolated...

  4. Systems and methods for the secretion of recombinant proteins in gram negative bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Withers, III, Sydnor T.; Dominguez, Miguel A.; DeLisa, Matthew P.; Haitjema, Charles H.

    2017-02-21

    Disclosed herein are systems and methods for producing recombinant proteins utilizing mutant E. coli strains containing expression vectors carrying nucleic acids encoding the proteins, and secretory signal sequences to direct the secretion of the proteins to the culture medium. Host cells transformed with the expression vectors are also provided.

  5. Recombinant protein production data after expression in the bacterium Escherichia coli

    Directory of Open Access Journals (Sweden)

    J. Enrique Cantu-Bustos

    2016-06-01

    Full Text Available Fusion proteins have become essential for the expression and purification of recombinant proteins in Escherichia coli. The metal-binding protein CusF has shown several features that make it an attractive fusion protein and affinity tag: "Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF" (Cantu-Bustos et al., 2016 [1]. Here we present accompanying data from protein expression experiments; we tested different protein tags, temperatures, expression times, cellular compartments, and concentrations of inducer in order to obtain soluble protein and low formation of inclusion bodies. Additionally, we present data from the purification of the green fluorescent protein (GFP tagged with CusF, using Ag(I metal affinity chromatography.

  6. An inducible expression system for high-level expression of recombinant proteins in slow growing mycobacteria.

    Science.gov (United States)

    Leotta, Lisa; Spratt, Joanne M; Kong, Carlyn U; Triccas, James A

    2015-09-01

    A novel protein expression vector utilising the inducible hspX promoter of Mycobacterium tuberculosis was constructed and evaluated in this study. High-level induction of three mycobacterial antigens, comprising up to 9% of bacterial sonicate, was demonstrated in recombinant Mycobacterium bovis BCG when grown under low-oxygen tension, which serves to enhance hspX promoter activity. Recombinant proteins were efficiently purified from bacterial lysates in a soluble form by virtue of a C-terminal 6-histidine tag. Purification of the immunodominant M. tuberculosis Ag85B antigen using this system resulted in a recombinant protein that stimulated significant IFN-γ release from Ag85B-reactive T cells generated after vaccination of mice with an Ag85B-expressing vaccine. Further, the M. tuberculosis L-alanine dehydrogenase (Ald) protein purified from recombinant BCG displayed strong enzymatic activity in recombinant form. This study demonstrated that high levels of native-like recombinant mycobacterial proteins can be produced in mycobacterial hosts, and this may aid the analysis of mycobacterial protein function and the development of new treatments. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Use of a protein engineering strategy to overcome limitations in the production of "Difficult to Express" recombinant proteins.

    Science.gov (United States)

    Hussain, Hirra; Fisher, David I; Abbott, W Mark; Roth, Robert G; Dickson, Alan J

    2017-10-01

    Certain recombinant proteins are deemed "difficult to express" in mammalian expression systems requiring significant cell and/or process engineering to abrogate expression bottlenecks. With increasing demand for the production of recombinant proteins in mammalian cells, low protein yields can have significant consequences for industrial processes. To investigate the molecular mechanisms that restrict expression of recombinant proteins, naturally secreted model proteins were analyzed from the tissue inhibitors of metalloproteinase (TIMP) protein family. In particular, TIMP-2 and TIMP-3 were subjected to detailed study. TIMP proteins share significant sequence homology (∼50% identity and ∼70% similarity in amino acid sequence). However, they show marked differences in secretion in mammalian expression systems despite this extensive sequence homology. Using these two proteins as models, this study characterized the molecular mechanisms responsible for poor recombinant protein production. Our results reveal that both TIMP-2 and TIMP-3 are detectable at mRNA and protein level within the cell but only TIMP-2 is secreted effectively into the extracellular medium. Analysis of protein localization and the nature of intracellular protein suggest TIMP-3 is severely limited in its post-translational processing. To overcome this challenge, modification of the TIMP-3 sequence to include a furin protease-cleavable pro-sequence resulted in secretion of the modified TIMP-3 protein, however, incomplete processing was observed. Based on the TIMP-3 data, the protein engineering approach was optimized and successfully applied in combination with cell engineering, the overexpression of furin, to another member of the TIMP protein family (the poorly expressed TIMP-4). Use of the described protein engineering strategy resulted in successful secretion of poorly (TIMP-4) and non-secreted (TIMP-3) targets, and presents a novel strategy to enhance the production of "difficult" recombinant

  8. Regulation of homologous recombination repair protein Rad51 by Ku70

    International Nuclear Information System (INIS)

    Du Liqing; Liu Qiang; Wang Yan; Xu Chang; Cao Jia; Fu Yue; Chen Fenghua; Fan Feiyue

    2013-01-01

    Objective: To explore the regulative effect of non-homologous end joining (NHEJ)protein Ku70 on homologous recombination repair protein Rad51, and to investigate the synergistic mechanism of homologous recombination repair in combination with NHEJ. Methods: Observed Rad51 protein expression after transfect Ku70 small interfering RNA or Ku70 plasmid DNA into tumor cells using Western blot. Results: Expression of Rad51 was obviously reduced after pretreated with Ku70 small interfering RNA. And with the increasing expression of Ku70 protein after transfection of Ku70 plasmid DNA PGCsi3.0-hKu70 into tumor cell lines, the Rad51 protein expression was increased. Conclusion: Ku70 protein has regulating effect on gene expression of Rad51, and it might participate in the collaboration between homologous recombination repair and NHEJ. (authors)

  9. Combinations of SPR and MS for Characterizations of Native and Recombinant Proteins in Cell Lysates

    DEFF Research Database (Denmark)

    Borch, Jonas; Roepstorff, Peter

    2006-01-01

    Surface plasmon resonance and mass spectrometry (SPR-MS) has been combined for quality check of recombinant 6xHis-tagged 14-3-3 proteins expressed in Escherichia coli. Lysates were injected over an SPR sensorchip with immobilized Ni2+ for SPR analysis of the specific Ni2+ binding response...... and stability. To validate the identity, intactness and homogeneity of the captured proteins were eluted for mass spectrometric analysis of intact molecular weight and peptide mass mapping. Additionally, the captured recombinant proteins were investigated for specific binding to known phosphorylated ligands...... of 14-3-3 proteins in order to test their activity. Specific binding of recombinant and native 14-3-3 proteins in complex mixtures to immobilized phosphopeptides and subsequent elution was also tested by SPR-MS. Ammonium sulfate precipitate fractions from lysates of E. coli expressing 14-3-3 protein...

  10. Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli

    DEFF Research Database (Denmark)

    Sørensen, Hans; Mortensen, Kim

    2005-01-01

    Pure, soluble and functional proteins are of high demand in modern biotechnology. Natural protein sources rarely meet the requirements for quantity, ease of isolation or price and hence recombinant technology is often the method of choice. Recombinant cell factories are constantly employed...... molecular tools available. In spite of all these qualities, expression of recombinant proteins with E. coli as the host often results in insoluble and/or nonfunctional proteins. Here we review new approaches to overcome these obstacles by strategies that focus on either controlled expression of target...... for the production of protein preparations bound for downstream purification and processing. Eschericia coli is a frequently used host, since it facilitates protein expression by its relative simplicity, its inexpensive and fast high density cultivation, the well known genetics and the large number of compatible...

  11. Exploiting translational coupling for the selection of cells producing toxic recombinant proteins from expression vectors.

    Science.gov (United States)

    Tagliavia, Marcello; Cuttitta, Angela

    2016-01-01

    High rates of plasmid instability are associated with the use of some expression vectors in Escherichia coli, resulting in the loss of recombinant protein expression. This is due to sequence alterations in vector promoter elements caused by the background expression of the cloned gene, which leads to the selection of fast-growing, plasmid-containing cells that do not express the target protein. This phenomenon, which is worsened when expressing toxic proteins, results in preparations containing very little or no recombinant protein, or even in clone loss; however, no methods to prevent loss of recombinant protein expression are currently available. We have exploited the phenomenon of translational coupling, a mechanism of prokaryotic gene expression regulation, in order to select cells containing plasmids still able to express recombinant proteins. Here we designed an expression vector in which the cloned gene and selection marker are co-expressed. Our approach allowed for the selection of the recombinant protein-expressing cells and proved effective even for clones encoding toxic proteins.

  12. The synthesis of recombinant membrane proteins in yeast for structural studies.

    Science.gov (United States)

    Routledge, Sarah J; Mikaliunaite, Lina; Patel, Anjana; Clare, Michelle; Cartwright, Stephanie P; Bawa, Zharain; Wilks, Martin D B; Low, Floren; Hardy, David; Rothnie, Alice J; Bill, Roslyn M

    2016-02-15

    Historically, recombinant membrane protein production has been a major challenge meaning that many fewer membrane protein structures have been published than those of soluble proteins. However, there has been a recent, almost exponential increase in the number of membrane protein structures being deposited in the Protein Data Bank. This suggests that empirical methods are now available that can ensure the required protein supply for these difficult targets. This review focuses on methods that are available for protein production in yeast, which is an important source of recombinant eukaryotic membrane proteins. We provide an overview of approaches to optimize the expression plasmid, host cell and culture conditions, as well as the extraction and purification of functional protein for crystallization trials in preparation for structural studies. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  13. Hormad1 mutation disrupts synaptonemal complex formation, recombination, and chromosome segregation in mammalian meiosis.

    Directory of Open Access Journals (Sweden)

    Yong-Hyun Shin

    2010-11-01

    Full Text Available Meiosis is unique to germ cells and essential for reproduction. During the first meiotic division, homologous chromosomes pair, recombine, and form chiasmata. The homologues connect via axial elements and numerous transverse filaments to form the synaptonemal complex. The synaptonemal complex is a critical component for chromosome pairing, segregation, and recombination. We previously identified a novel germ cell-specific HORMA domain encoding gene, Hormad1, a member of the synaptonemal complex and a mammalian counterpart to the yeast meiotic HORMA domain protein Hop1. Hormad1 is essential for mammalian gametogenesis as knockout male and female mice are infertile. Hormad1 deficient (Hormad1(-/ (- testes exhibit meiotic arrest in the early pachytene stage, and synaptonemal complexes cannot be visualized by electron microscopy. Hormad1 deficiency does not affect localization of other synaptonemal complex proteins, SYCP2 and SYCP3, but disrupts homologous chromosome pairing. Double stranded break formation and early recombination events are disrupted in Hormad1(-/ (- testes and ovaries as shown by the drastic decrease in the γH2AX, DMC1, RAD51, and RPA foci. HORMAD1 co-localizes with γH2AX to the sex body during pachytene. BRCA1, ATR, and γH2AX co-localize to the sex body and participate in meiotic sex chromosome inactivation and transcriptional silencing. Hormad1 deficiency abolishes γH2AX, ATR, and BRCA1 localization to the sex chromosomes and causes transcriptional de-repression on the X chromosome. Unlike testes, Hormad1(-/ (- ovaries have seemingly normal ovarian folliculogenesis after puberty. However, embryos generated from Hormad1(-/ (- oocytes are hyper- and hypodiploid at the 2 cell and 8 cell stage, and they arrest at the blastocyst stage. HORMAD1 is therefore a critical component of the synaptonemal complex that affects synapsis, recombination, and meiotic sex chromosome inactivation and transcriptional silencing.

  14. Expression and purification of recombinant proteins in Escherichia coli tagged with a small metal-binding protein from Nitrosomonas europaea.

    Science.gov (United States)

    Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Zarate, Xristo

    2016-02-01

    Escherichia coli is still the preferred organism for large-scale production of recombinant proteins. The use of fusion proteins has helped considerably in enhancing the solubility of heterologous proteins and their purification with affinity chromatography. Here, the use of a small metal-binding protein (SmbP) from Nitrosomonas europaea is described as a new fusion protein for protein expression and purification in E. coli. Fluorescent proteins tagged at the N-terminal with SmbP showed high levels of solubility, compared with those of maltose-binding protein and glutathione S-transferase, and low formation of inclusion bodies. Using commercially available IMAC resins charged with Ni(II), highly pure recombinant proteins were obtained after just one chromatography step. Proteins may be purified from the periplasm of E. coli if SmbP contains the signal sequence at the N-terminal. After removal of the SmbP tag from the protein of interest, high-yields are obtained since SmbP is a protein of just 9.9 kDa. The results here obtained suggest that SmbP is a good alternative as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Production of recombinant dengue non-structural 1 (NS1) proteins from clinical virus isolates.

    Science.gov (United States)

    Yohan, Benediktus; Wardhani, Puspa; Aryati; Trimarsanto, Hidayat; Sasmono, R Tedjo

    2017-01-01

    Dengue is a febrile disease caused by infection of dengue virus (DENV). Early diagnosis of dengue infection is important for better management of the disease. The DENV Non-Structural Protein 1 (NS1) antigen has been routinely used for the early dengue detection. In dengue epidemic countries such as Indonesia, clinicians are increasingly relying on the NS1 detection for confirmation of dengue infection. Various NS1 diagnostic tests are commercially available, however different sensitivities and specificities were observed in various settings. This study was aimed to generate dengue NS1 recombinant protein for the development of dengue diagnostic tests. Four Indonesian DENV isolates were used as the source of the NS1 gene cloning, expression, and purification in bacterial expression system. Recombinant NS1 proteins were successfully purified and their antigenicities were assessed. Immunization of mice with recombinant proteins observed the immunogenicity of the NS1 protein. The generated recombinant proteins can be potentially used in the development of NS1 diagnostic test. With minimal modifications, this method can be used for producing NS1 recombinant proteins from isolates obtained from other geographical regions. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Binding of recombinant apolipoprotein(a) to extracellular matrix proteins

    NARCIS (Netherlands)

    van der Hoek, Y. Y.; Sangrar, W.; Côté, G. P.; Kastelein, J. J.; Koschinsky, M. L.

    1994-01-01

    Elevated levels of lipoprotein(a), which consists of apolipoprotein(a) [apo(a)] covalently linked to a low-density lipoprotein-like moiety, is an independent risk factor for the development of atherosclerosis. We show that a recombinant form of apo(a) [r-apo(a)] binds strongly to fibronectin and

  17. Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein

    OpenAIRE

    Bertagnoli, Stéphane; Gelfi, Jacqueline; Le Gall, Ghislaine; Boilletot, Eric; Vautherot, Jean-François; Rasschaert, Denis; Laurent, Sylvie; Petit, Frédérique; Boucraut-Baralon, Corine; Milon, Alain

    1996-01-01

    Two myxoma virus-rabbit hemorrhagic disease virus (RHDV) recombinant viruses were constructed with the SG33 strain of myxoma virus to protect rabbits against myxomatosis and rabbit viral hemorrhagic disease. These recombinant viruses expressed the RHDV capsid protein (VP60). The recombinant protein, which is 60 kDa in size, was antigenic, as revealed by its reaction in immunoprecipitation with antibodies raised against RHDV. Both recombinant viruses induced high levels of RHDV- and myxoma vir...

  18. Plastoglobules: a new address for targeting recombinant proteins in the chloroplast

    Directory of Open Access Journals (Sweden)

    Kessler Felix

    2007-01-01

    Full Text Available Abstract Background The potential of transgenic plants for cost-effective production of pharmaceutical molecules is now becoming apparent. Plants have the advantage over established fermentation systems (bacterial, yeast or animal cell cultures to circumvent the risk of pathogen contamination, to be amenable to large scaling up and to necessitate only established farming procedures. Chloroplasts have proven a useful cellular compartment for protein accumulation owing to their large size and number, as well as the possibility for organellar transformation. They therefore represent the targeting destination of choice for recombinant proteins in leaf crops such as tobacco. Extraction and purification of recombinant proteins from leaf material contribute to a large extent to the production costs. Developing new strategies facilitating these processes is therefore necessary. Results Here, we evaluated plastoglobule lipoprotein particles as a new subchloroplastic destination for recombinant proteins. The yellow fluorescent protein as a trackable cargo was targeted to plastoglobules when fused to plastoglobulin 34 (PGL34 as the carrier. Similar to adipocyte differentiation related protein (ADRP in animal cells, most of the protein sequence of PGL34 was necessary for targeting to lipid bodies. The recombinant protein was efficiently enriched in plastoglobules isolated by simple flotation centrifugation. The viability of plants overproducing the recombinant protein was not affected, indicating that plastoglobule targeting did not significantly impair photosynthesis or sugar metabolism. Conclusion Our data identify plastoglobules as a new targeting destination for recombinant protein in leaf crops. The wide-spread presence of plastoglobules and plastoglobulins in crop species promises applications comparable to those of transgenic oilbody-oleosin technology in molecular farming.

  19. Ex-vivo assessment of chronic toxicity of low levels of cadmium on testicular meiotic cells

    Energy Technology Data Exchange (ETDEWEB)

    Geoffroy-Siraudin, Cendrine [Aix-Marseille Univ, UMR CNRS IMBE 7263, FR 3098 ECCOREV, 13005, Marseille (France); Laboratoire de Biologie de la Reproduction, AP-HM, Hôpital de la Conception, 147, Boulevard Baille, 13385 Marseille cedex 5 (France); Perrard, Marie-Hélène [Institut de Génomique Fonctionnelle de Lyon, UMR 5242 CNRS INRA Ecole Normale Supérieure de Lyon 1, 46 allée d' Italie, F-69364 Lyon Cedex 07 (France); Ghalamoun-Slaimi, Rahma [Aix-Marseille Univ, UMR CNRS IMBE 7263, FR 3098 ECCOREV, 13005, Marseille (France); Laboratoire de Biologie de la Reproduction, AP-HM, Hôpital de la Conception, 147, Boulevard Baille, 13385 Marseille cedex 5 (France); Ali, Sazan [Aix-Marseille Univ, UMR CNRS IMBE 7263, FR 3098 ECCOREV, 13005, Marseille (France); Chaspoul, Florence [Aix-Marseille Univ, UMR CNRS IMBE 7263, FR 3098 ECCOREV, 13005, Marseille (France); Unité de Chimie-Physique, Faculté de Pharmacie 13005, Marseille (France); Lanteaume, André [Aix-Marseille Univ, UMR CNRS IMBE 7263, FR 3098 ECCOREV, 13005, Marseille (France); Achard, Vincent [Laboratoire de Biologie de la Reproduction, AP-HM, Hôpital de la Conception, 147, Boulevard Baille, 13385 Marseille cedex 5 (France); Gallice, Philippe [Aix-Marseille Univ, UMR CNRS IMBE 7263, FR 3098 ECCOREV, 13005, Marseille (France); Unité de Chimie-Physique, Faculté de Pharmacie 13005, Marseille (France); Durand, Philippe [Institut de Génomique Fonctionnelle de Lyon, UMR 5242 CNRS INRA Ecole Normale Supérieure de Lyon 1, 46 allée d' Italie, F-69364 Lyon Cedex 07 (France); and others

    2012-08-01

    Using a validated model of culture of rat seminiferous tubules, we assessed the effects of 0.1, 1 and 10 μg/L cadmium (Cd) on spermatogenic cells over a 2‐week culture period. With concentrations of 1 and 10 μg/L in the culture medium, the Cd concentration in the cells, determined by ICP-MS, increased with concentration in the medium and the day of culture. Flow cytometric analysis enabled us to evaluate changes in the number of Sertoli cells and germ cells during the culture period. The number of Sertoli cells did not appear to be affected by Cd. By contrast, spermatogonia and meiotic cells were decreased by 1 and 10 μg/L Cd in a time and dose dependent manner. Stage distribution of the meiotic prophase I and qualitative study of the synaptonemal complexes (SC) at the pachytene stage were performed by immunocytochemistry with an anti SCP3 antibody. Cd caused a time-and-dose-dependent increase of total abnormalities, of fragmented SC and of asynapsis from concentration of 0.1 μg/L. Additionally, we observed a new SC abnormality, the “motheaten” SC. This abnormality is frequently associated with asynapsis and SC widening which increased with both the Cd concentration and the duration of exposure. This abnormality suggests that Cd disrupts the structure and function of proteins involved in pairing and/or meiotic recombination. These results show that Cd induces dose-and-time-dependent alterations of the meiotic process of spermatogenesis ex-vivo, and that the lowest metal concentration, which induces an adverse effect, may vary with the cell parameter studied. -- Highlights: ► Cadmium induces ex-vivo severe time- and dose-dependent germ cell abnormalities. ► Cadmium at very low concentration (0.1 µg/l) induces synaptonemal complex abnormalities. ► The lowest concentration inducing adverse effect varied with the cell parameter studied. ► Cadmium alters proteins involved in pairing and recombination. ► Cadmium leads to achiasmate univalents and

  20. Cytological studies of human meiosis: sex-specific differences in recombination originate at, or prior to, establishment of double-strand breaks.

    Directory of Open Access Journals (Sweden)

    Jennifer R Gruhn

    Full Text Available Meiotic recombination is sexually dimorphic in most mammalian species, including humans, but the basis for the male:female differences remains unclear. In the present study, we used cytological methodology to directly compare recombination levels between human males and females, and to examine possible sex-specific differences in upstream events of double-strand break (DSB formation and synaptic initiation. Specifically, we utilized the DNA mismatch repair protein MLH1 as a marker of recombination events, the RecA homologue RAD51 as a surrogate for DSBs, and the synaptonemal complex proteins SYCP3 and/or SYCP1 to examine synapsis between homologs. Consistent with linkage studies, genome-wide recombination levels were higher in females than in males, and the placement of exchanges varied between the sexes. Subsequent analyses of DSBs and synaptic initiation sites indicated similar male:female differences, providing strong evidence that sex-specific differences in recombination rates are established at or before the formation of meiotic DSBs. We then asked whether these differences might be linked to variation in the organization of the meiotic axis and/or axis-associated DNA and, indeed, we observed striking male:female differences in synaptonemal complex (SC length and DNA loop size. Taken together, our observations suggest that sex specific differences in recombination in humans may derive from chromatin differences established prior to the onset of the recombination pathway.

  1. Increasing the production yield of recombinant protein in transgenic seeds by expanding the deposition space within the intracellular compartment

    OpenAIRE

    Takaiwa, Fumio

    2013-01-01

    Seeds must maintain a constant level of nitrogen in order to germinate. When recombinant proteins are produced while endogenous seed protein expression is suppressed, the production levels of the foreign proteins increase to compensate for the decreased synthesis of endogenous proteins. Thus, exchanging the production of endogenous seed proteins for that of foreign proteins is a promising approach to increase the yield of foreign recombinant proteins. Providing a space for the deposition of r...

  2. Isolation of recombinant antibodies directed against surface proteins of Clostridium difficile.

    Science.gov (United States)

    Shirvan, Ali Nazari; Aitken, Robert

    2016-01-01

    Clostridium difficile has emerged as an increasingly important nosocomial pathogen and the prime causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis in humans. In addition to toxins A and B, immunological studies using antisera from patients infected with C. difficile have shown that a number of other bacterial factors contribute to the pathogenesis, including surface proteins, which are responsible for adhesion, motility and other interactions with the human host. In this study, various clostridial targets, including FliC, FliD and cell wall protein 66, were expressed and purified. Phage antibody display yielded a large panel of specific recombinant antibodies, which were expressed, purified and characterised. Reactions of the recombinant antibodies with their targets were detected by enzyme-linked immunosorbent assay; and Western blotting suggested that linear rather than conformational epitopes were recognised. Binding of the recombinant antibodies to surface-layer proteins and their components showed strain specificity, with good recognition of proteins from C. difficile 630. However, no reaction was observed for strain R20291-a representative of the 027 ribotype. Binding of the recombinant antibodies to C. difficile M120 extracts indicated that a component of a surface-layer protein of this strain might possess immunoglobulin-binding activities. The recombinant antibodies against FliC and FliD proteins were able to inhibit bacterial motility. Copyright © 2016. Published by Elsevier Editora Ltda.

  3. Photosynthetic biomanufacturing in green algae; production of recombinant proteins for industrial, nutritional, and medical uses.

    Science.gov (United States)

    Rasala, Beth A; Mayfield, Stephen P

    2015-03-01

    Recombinant proteins are widely used for industrial, nutritional, and medical applications. Green microalgae have attracted considerable attention recently as a biomanufacturing platform for the production of recombinant proteins for a number of reasons. These photosynthetic eukaryotic microorganisms are safe, scalable, easy to genetically modify through transformation, mutagenesis, or breeding, and inexpensive to grow. Many microalgae species are genetically transformable, but the green alga Chlamydomonas reinhardtii is the most widely used host for recombinant protein expression. An extensive suite of molecular genetic tools has been developed for C. reinhardtii over the last 25 years, including a fully sequenced genome, well-established methods for transformation, mutagenesis and breeding, and transformation vectors for high levels of recombinant protein accumulation and secretion. Here, we review recent successes in the development of C. reinhardtii as a biomanufacturing host for recombinant proteins, including antibodies and immunotoxins, hormones, industrial enzymes, an orally-active colostral protein for gastrointestinal health, and subunit vaccines. In addition, we review the biomanufacturing potential of other green algae from the genera Dunaliella and Chlorella.

  4. Expression and the antigenicity of recombinant coat proteins of tungro viruses expressed in Escherichia coli.

    Science.gov (United States)

    Yee, Siew Fung; Chu, Chia Huay; Poili, Evenni; Sum, Magdline Sia Henry

    2017-02-01

    Rice tungro disease (RTD) is a recurring disease affecting rice farming especially in the South and Southeast Asia. The disease is commonly diagnosed by visual observation of the symptoms on diseased plants in paddy fields and by polymerase chain reaction (PCR). However, visual observation is unreliable and PCR can be costly. High-throughput as well as relatively cheap detection methods are important for RTD management for screening large number of samples. Due to this, detection by serological assays such as immunoblotting assays and enzyme-linked immunosorbent assay are preferred. However, these serological assays are limited by lack of continuous supply of antibodies as reagents due to the difficulty in preparing sufficient purified virions as antigens. This study aimed to generate and evaluate the reactivity of the recombinant coat proteins of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV) as alternative antigens to generate antibodies. The genes encoding the coat proteins of both viruses, RTBV (CP), and RTSV (CP1, CP2 and CP3) were cloned and expressed as recombinant fusion proteins in Escherichia coli. All of the recombinant fusion proteins, with the exception of the recombinant fusion protein of the CP2 of RTSV, were reactive against our in-house anti-tungro rabbit serum. In conclusion, our study showed the potential use of the recombinant fusion coat proteins of the tungro viruses as alternative antigens for production of antibodies for diagnostic purposes. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Capillary gel electrophoresis for the quantification and purity determination of recombinant proteins in inclusion bodies.

    Science.gov (United States)

    Espinosa-de la Garza, Carlos E; Perdomo-Abúndez, Francisco C; Campos-García, Víctor R; Pérez, Néstor O; Flores-Ortiz, Luis F; Medina-Rivero, Emilio

    2013-09-01

    In this work, a high-resolution CGE method for quantification and purity determination of recombinant proteins was developed, involving a single-component inclusion bodies (IBs) solubilization solution. Different recombinant proteins expressed as IBs were used to show method capabilities, using recombinant interferon-β 1b as the model protein for method validation. Method linearity was verified in the range from 0.05 to 0.40 mg/mL and a determination coefficient (r(2) ) of 0.99 was obtained. The LOQs and LODs were 0.018 and 0.006 mg/mL, respectively. RSD for protein content repeatability test was 2.29%. In addition, RSD for protein purity repeatability test was 4.24%. Method accuracy was higher than 90%. Specificity was confirmed, as the method was able to separate recombinant interferon-β 1b monomer from other aggregates and impurities. Sample content and purity was demonstrated to be stable for up to 48 h. Overall, this method is suitable for the analysis of recombinant proteins in IBs according to the attributes established on the International Conference for Harmonization guidelines. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Recombining without Hotspots: A Comprehensive Evolutionary Portrait of Recombination in Two Closely Related Species of Drosophila

    Science.gov (United States)

    Smukowski Heil, Caiti S.; Ellison, Chris; Dubin, Matthew; Noor, Mohamed A.F.

    2015-01-01

    Meiotic recombination rate varies across the genome within and between individuals, populations, and species in virtually all taxa studied. In almost every species, this variation takes the form of discrete recombination hotspots, determined in some mammals by a protein called PRDM9. Hotspots and their determinants have a profound effect on the genomic landscape, and share certain features that extend across the tree of life. Drosophila, in contrast, are anomalous in their absence of hotspots, PRDM9, and other species-specific differences in the determination of recombination. To better understand the evolution of meiosis and general patterns of recombination across diverse taxa, we present a truly comprehensive portrait of recombination across time, combining recently published cross-based contemporary recombination estimates from each of two sister species with newly obtained linkage-disequilibrium-based historic estimates of recombination from both of these species. Using Drosophila pseudoobscura and Drosophila miranda as a model system, we compare recombination rate between species at multiple scales, and we suggest that Drosophila replicate the pattern seen in human–chimpanzee in which recombination rate is conserved at broad scales. We also find evidence of a species-wide recombination modifier(s), resulting in both a present and historic genome-wide elevation of recombination rates in D. miranda, and identify broad scale effects on recombination from the presence of an inversion. Finally, we reveal an unprecedented view of the distribution of recombination in D. pseudoobscura, illustrating patterns of linked selection and where recombination is taking place. Overall, by combining these estimation approaches, we highlight key similarities and differences in recombination between Drosophila and other organisms. PMID:26430062

  7. Recombining without Hotspots: A Comprehensive Evolutionary Portrait of Recombination in Two Closely Related Species of Drosophila.

    Science.gov (United States)

    Smukowski Heil, Caiti S; Ellison, Chris; Dubin, Matthew; Noor, Mohamed A F

    2015-10-01

    Meiotic recombination rate varies across the genome within and between individuals, populations, and species in virtually all taxa studied. In almost every species, this variation takes the form of discrete recombination hotspots, determined in some mammals by a protein called PRDM9. Hotspots and their determinants have a profound effect on the genomic landscape, and share certain features that extend across the tree of life. Drosophila, in contrast, are anomalous in their absence of hotspots, PRDM9, and other species-specific differences in the determination of recombination. To better understand the evolution of meiosis and general patterns of recombination across diverse taxa, we present a truly comprehensive portrait of recombination across time, combining recently published cross-based contemporary recombination estimates from each of two sister species with newly obtained linkage-disequilibrium-based historic estimates of recombination from both of these species. Using Drosophila pseudoobscura and Drosophila miranda as a model system, we compare recombination rate between species at multiple scales, and we suggest that Drosophila replicate the pattern seen in human-chimpanzee in which recombination rate is conserved at broad scales. We also find evidence of a species-wide recombination modifier(s), resulting in both a present and historic genome-wide elevation of recombination rates in D. miranda, and identify broad scale effects on recombination from the presence of an inversion. Finally, we reveal an unprecedented view of the distribution of recombination in D. pseudoobscura, illustrating patterns of linked selection and where recombination is taking place. Overall, by combining these estimation approaches, we highlight key similarities and differences in recombination between Drosophila and other organisms. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  8. Recombinant fusion protein of albumin-retinol binding protein inactivates stellate cells

    International Nuclear Information System (INIS)

    Choi, Soyoung; Park, Sangeun; Kim, Suhyun; Lim, Chaeseung; Kim, Jungho; Cha, Dae Ryong; Oh, Junseo

    2012-01-01

    Highlights: ► We designed novel recombinant albumin-RBP fusion proteins. ► Expression of fusion proteins inactivates pancreatic stellate cells (PSCs). ► Fusion proteins are successfully internalized into and inactivate PSCs. ► RBP moiety mediates cell specific uptake of fusion protein. -- Abstract: Quiescent pancreatic- (PSCs) and hepatic- (HSCs) stellate cells store vitamin A (retinol) in lipid droplets via retinol binding protein (RBP) receptor and, when activated by profibrogenic stimuli, they transform into myofibroblast-like cells which play a key role in the fibrogenesis. Despite extensive investigations, there is, however, currently no appropriate therapy available for tissue fibrosis. We previously showed that the expression of albumin, composed of three homologous domains (I–III), inhibits stellate cell activation, which requires its high-affinity fatty acid-binding sites asymmetrically distributed in domain I and III. To attain stellate cell-specific uptake, albumin (domain I/III) was coupled to RBP; RBP-albumin domain III (R-III) and albumin domain I -RBP-albumin III (I-R-III). To assess the biological activity of fusion proteins, cultured PSCs were used. Like wild type albumin, expression of R-III or I-R-III in PSCs after passage 2 (activated PSCs) induced phenotypic reversal from activated to fat-storing cells. On the other hand, R-III and I-R-III, but not albumin, secreted from transfected 293 cells were successfully internalized into and inactivated PSCs. FPLC-purified R-III was found to be internalized into PSCs via caveolae-mediated endocytosis, and its efficient cellular uptake was also observed in HSCs and podocytes among several cell lines tested. Moreover, tissue distribution of intravenously injected R-III was closely similar to that of RBP. Therefore, our data suggest that albumin-RBP fusion protein comprises of stellate cell inactivation-inducing moiety and targeting moiety, which may lead to the development of effective anti

  9. Effect of sex, age, and breed on genetic recombination features in cattle

    Science.gov (United States)

    Meiotic recombination is a fundamental biological process which generates genetic diversity, affects fertility, and influences evolvability. Here we investigate the roles of sex, age, and breed in cattle recombination features, including recombination rate, location and crossover interference. Usin...

  10. Rapid production of functionalized recombinant proteins: marrying ligation independent cloning and in vitro protein ligation.

    Science.gov (United States)

    Kushnir, Susanna; Marsac, Yoann; Breitling, Reinhard; Granovsky, Igor; Brok-Volchanskaya, Vera; Goody, Roger S; Becker, Christian F W; Alexandrov, Kirill

    2006-01-01

    Functional genomics and proteomics have been very active fields since the sequencing of several genomes was completed. To assign a physiological role to the newly discovered coding genes with unknown function, new generic methods for protein production, purification, and targeted functionalization are needed. This work presents a new vector, pCYSLIC, that allows rapid generation of Escherichia coli expression constructs via ligation-independent cloning (LIC). The vector is designed to facilitate protein purification by either Ni-NTA or GSH affinity chromatography. Subsequent proteolytic removal of affinity tags liberates an N-terminal cysteine residue that is then used for covalent modification of the target protein with different biophysical probes via protein ligation. The described system has been tested on 36 mammalian Rab GTPases, and it was demonstrated that recombinant GTPases produced with pCYSLIC could be efficiently modified with fluorescein or biotin in vitro. Finally, LIC was compared with the recently developed In-Fusion cloning method, and it was demonstrated that In-Fusion provides superior flexibility in choice of expression vector. By the application of In-Fusion cloning Cys-Rab6A GTPase with an N-terminal cysteine residue was generated employing unmodified pET30a vector and TVMV protease.

  11. Choreography of recombination proteins during the DNA damage response

    DEFF Research Database (Denmark)

    Lisby, Michael; Rothstein, Rodney

    2009-01-01

    Genome integrity is frequently challenged by DNA lesions from both endogenous and exogenous sources. A single DNA double-strand break (DSB) is lethal if unrepaired and may lead to loss of heterozygosity, mutations, deletions, genomic rearrangements and chromosome loss if repaired improperly. Such...... research. Here we review the cell biological response to DSBs in mitotically growing cells with an emphasis on homologous recombination pathways in yeast Saccharomyces cerevisiae and in mammalian cells....

  12. Transphosphorylation of E. coli proteins during production of recombinant protein kinases provides a robust system to characterize kinase specificity

    Science.gov (United States)

    Protein kinase specificity is of fundamental importance to pathway regulation and signal transduction. Here, we report a convenient system to monitor the activity and specificity of recombinant protein kinases expressed in E.coli. We apply this to the study of the cytoplasmic domain of the plant rec...

  13. Expression and Characterisation of Recombinant Rhodocyclus tenuis High Potential Iron-Sulphur Protein

    DEFF Research Database (Denmark)

    Caspersen, Michael Bjerg; Bennet, K.; Christensen, Hans Erik Mølager

    2000-01-01

    The high potential iron-sulfur protein (HiPIP) from Rhodocyclus tenuis strain 2761 has been overproduced in Escherichia coli from its structural gene, purified to apparent homogeneity, and then characterized by an array of methods. UV-visible spectra of the reduced and oxidized recombinant protein...

  14. The recombinant 120-kilodalton protein of Ehrlichia chaffeensis, a potential diagnostic tool.

    OpenAIRE

    Yu, X J; Crocquet-Valdes, P; Cullman, L C; Walker, D H

    1996-01-01

    DNA encoding two repeat units of 120-kDa protein of Ehrlichia chaffeensis was cloned into the expression vector pGEX and expressed in Escherichia coli. The sensitivity and specificity of a dot blot assay for detection of human antibodies with the recombinant protein were 86 and 100%, respectively, compared with an indirect immunofluorescence assay.

  15. Chapter 15. transforming lepidopteran insect cells for continuous recombinant protein expression

    Science.gov (United States)

    The baculovirus expression vector system (BEVS) is widely used to produce large quantities of recombinant proteins. However, yields of extracellular and membrane-bound proteins obtained with this system often are very low, possibly due to the adverse effects of baculovirus infection on the host ins...

  16. Evolved Lactococcus lactis Strains for Enhanced Expression of Recombinant Membrane Proteins

    NARCIS (Netherlands)

    Martinez Linares, Daniel; Geertsma, Eric R.; Poolman, Bert

    2010-01-01

    The production of complex multidomain (membrane) proteins is a major hurdle in structural genomics and a generic approach for optimizing membrane protein expression is still lacking. We have devised a selection method to isolate mutant strains with improved functional expression of recombinant

  17. Gene and process level modulation to overcome the bottlenecks of recombinant proteins expression in Pichia pastoris.

    Science.gov (United States)

    Prabhu, Ashish A; Boro, Bibari; Bharali, Biju; Chakraborty, Shuchishloka; Dasu, V Venkata

    2018-03-28

    Process development involving system metabolic engineering and bioprocess engineering has become one of the major thrust for the development of therapeutic proteins or enzymes. Pichia pastoris has emerged as a prominent host for the production of therapeutic protein or enzymes. Despite of producing high protein titers, various cellular and process level bottlenecks hinders the expression of recombinant proteins in P. pastoris. In the present review, we have summarized the recent developments in the expression of foreign proteins in P. pastoris. Further, we have discussed various cellular engineering strategies which include codon optimization, pathway engineering, signal peptide processing, development of protease deficient strain and glyco-engineered strains for the high yield protein secretion of recombinant protein. Bioprocess development of recombinant proteins in large scale bioreactor including medium optimization, optimum feeding strategy and co-substrate feeding in fed batch as well as continuous cultivation have been described. The recent advances in system and synthetic biology studies including metabolic flux analysis in understanding the phenotypic characteristics of recombinant Pichia and genome editing with CRISPR-CAS system have also been summarized. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  18. Recombinant Expression Screening of P. aeruginosa Bacterial Inner Membrane Proteins

    Directory of Open Access Journals (Sweden)

    Jeffery Constance J

    2010-11-01

    Full Text Available Abstract Background Transmembrane proteins (TM proteins make up 25% of all proteins and play key roles in many diseases and normal physiological processes. However, much less is known about their structures and molecular mechanisms than for soluble proteins. Problems in expression, solubilization, purification, and crystallization cause bottlenecks in the characterization of TM proteins. This project addressed the need for improved methods for obtaining sufficient amounts of TM proteins for determining their structures and molecular mechanisms. Results Plasmid clones were obtained that encode eighty-seven transmembrane proteins with varying physical characteristics, for example, the number of predicted transmembrane helices, molecular weight, and grand average hydrophobicity (GRAVY. All the target proteins were from P. aeruginosa, a gram negative bacterial opportunistic pathogen that causes serious lung infections in people with cystic fibrosis. The relative expression levels of the transmembrane proteins were measured under several culture growth conditions. The use of E. coli strains, a T7 promoter, and a 6-histidine C-terminal affinity tag resulted in the expression of 61 out of 87 test proteins (70%. In this study, proteins with a higher grand average hydrophobicity and more transmembrane helices were expressed less well than less hydrophobic proteins with fewer transmembrane helices. Conclusions In this study, factors related to overall hydrophobicity and the number of predicted transmembrane helices correlated with the relative expression levels of the target proteins. Identifying physical characteristics that correlate with protein expression might aid in selecting the "low hanging fruit", or proteins that can be expressed to sufficient levels using an E. coli expression system. The use of other expression strategies or host species might be needed for sufficient levels of expression of transmembrane proteins with other physical

  19. Prediction of recombinant protein overexpression in Escherichia coli using a machine learning based model (RPOLP).

    Science.gov (United States)

    Habibi, Narjeskhatoon; Norouzi, Alireza; Mohd Hashim, Siti Z; Shamsir, Mohd Shahir; Samian, Razip

    2015-11-01

    Recombinant protein overexpression, an important biotechnological process, is ruled by complex biological rules which are mostly unknown, is in need of an intelligent algorithm so as to avoid resource-intensive lab-based trial and error experiments in order to determine the expression level of the recombinant protein. The purpose of this study is to propose a predictive model to estimate the level of recombinant protein overexpression for the first time in the literature using a machine learning approach based on the sequence, expression vector, and expression host. The expression host was confined to Escherichia coli which is the most popular bacterial host to overexpress recombinant proteins. To provide a handle to the problem, the overexpression level was categorized as low, medium and high. A set of features which were likely to affect the overexpression level was generated based on the known facts (e.g. gene length) and knowledge gathered from related literature. Then, a representative sub-set of features generated in the previous objective was determined using feature selection techniques. Finally a predictive model was developed using random forest classifier which was able to adequately classify the multi-class imbalanced small dataset constructed. The result showed that the predictive model provided a promising accuracy of 80% on average, in estimating the overexpression level of a recombinant protein. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Hydroxyapatite coating on the titanium substrate modulated by a recombinant collagen-like protein

    International Nuclear Information System (INIS)

    Pan Mingli; Kong Xiangdong; Cai Yurong; Yao Juming

    2011-01-01

    Research highlights: → Hydroxyapatite was deposited on alkali-heat treated Ti substrate by immersing in 1.5 x SBF solution containing the recombinant collagen-like protein. → The recombinant collagen-like protein accelerated the preferential nucleation and growth of hydroxyapatite along c axis on the Ti substrate. → Hydroxyapatite-collagen composite on the Ti substrate promoted the attachment, subsequently proliferation and differentiation of MG-63 cells. - Abstract: Plenty of techniques have been developed to modify the surface character of titanium (Ti) and its alloys in order to realize their biological bond to natural bone. In this work, a biomimetic process was employed to form a hydroxyapatite (HAp) coating on the alkali-heat treated Ti substrate in 1.5 times simulated body fluid (1.5 x SBF) with the addition of a recombinant collagen-like protein. The coating was characterized using SEM-EDX, FESEM, and XRD. Results showed that the recombinant collagen-like protein could accelerate the preferential nucleation and directional growth along c axis of HAp on the pretreated Ti substrates. The investigation of in vitro cell cultivation showed that the existence of recombinant collagen-like protein in coating could improve the initial cell adhesion, proliferation and differentiation of MG-63 cells, which implied the materials possessed excellent biocompatibility and had a wide potential in biomedical application.

  1. Hydroxyapatite coating on the titanium substrate modulated by a recombinant collagen-like protein

    Energy Technology Data Exchange (ETDEWEB)

    Pan Mingli [Key Laboratory of Advanced Textile Materials and Manufacturing Technology of Ministry of Education, College of Materials and Textile, Zhejiang Sci-Tech University, Hangzhou 310018 (China); Kong Xiangdong [College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018 (China); Cai Yurong [Key Laboratory of Advanced Textile Materials and Manufacturing Technology of Ministry of Education, College of Materials and Textile, Zhejiang Sci-Tech University, Hangzhou 310018 (China); Yao Juming, E-mail: yaoj@zstu.edu.cn [Key Laboratory of Advanced Textile Materials and Manufacturing Technology of Ministry of Education, College of Materials and Textile, Zhejiang Sci-Tech University, Hangzhou 310018 (China)

    2011-04-15

    Research highlights: {yields} Hydroxyapatite was deposited on alkali-heat treated Ti substrate by immersing in 1.5 x SBF solution containing the recombinant collagen-like protein. {yields} The recombinant collagen-like protein accelerated the preferential nucleation and growth of hydroxyapatite along c axis on the Ti substrate. {yields} Hydroxyapatite-collagen composite on the Ti substrate promoted the attachment, subsequently proliferation and differentiation of MG-63 cells. - Abstract: Plenty of techniques have been developed to modify the surface character of titanium (Ti) and its alloys in order to realize their biological bond to natural bone. In this work, a biomimetic process was employed to form a hydroxyapatite (HAp) coating on the alkali-heat treated Ti substrate in 1.5 times simulated body fluid (1.5 x SBF) with the addition of a recombinant collagen-like protein. The coating was characterized using SEM-EDX, FESEM, and XRD. Results showed that the recombinant collagen-like protein could accelerate the preferential nucleation and directional growth along c axis of HAp on the pretreated Ti substrates. The investigation of in vitro cell cultivation showed that the existence of recombinant collagen-like protein in coating could improve the initial cell adhesion, proliferation and differentiation of MG-63 cells, which implied the materials possessed excellent biocompatibility and had a wide potential in biomedical application.

  2. Indirect Enzyme-Linked Immunosorbent Assay for Detection of Immunoglobulin G Reactive with a Recombinant Protein Expressed from the Gene Encoding the 116-Kilodalton Protein of Mycoplasma pneumoniae

    OpenAIRE

    Duffy, Michael F.; Whithear, Kevin G.; Noormohammadi, Amir H.; Markham, Philip F.; Catton, Michael; Leydon, Jennie; Browning, Glenn F.

    1999-01-01

    Serology remains the method of choice for laboratory diagnosis of Mycoplasma pneumoniae infection. Currently available serological tests employ complex cellular fractions of M. pneumoniae as antigen. To improve the specificity of M. pneumoniae diagnosis, a recombinant protein was assessed as a serodiagnostic reagent. A panel of recombinant proteins were expressed from a cloned M. pneumoniae gene that encodes a 116-kDa surface protein antigen. The recombinant proteins were assessed for reactiv...

  3. A Rapid Method for Determining the Concentration of Recombinant Protein Secreted from Pichia pastoris

    International Nuclear Information System (INIS)

    Sun, L W; Zhao, Y; Jiang, R; Song, Y; Feng, H; Feng, K; Niu, L P; Qi, C

    2011-01-01

    Pichia secretive expression system is one of powerful eukaryotic expression systems in genetic engineering, which is especially suitable for industrial utilization. Because of the low concentration of the target protein in initial experiment, the methods and conditions for expression of the target protein should be optimized according to the protein yield repetitively. It is necessary to set up a rapid, simple and convenient analysis method for protein expression levels instead of the generally used method such as ultrafiltration, purification, dialysis, lyophilization and so on. In this paper, acetone precipitation method was chosen to concentrate the recombinant protein firstly after comparing with four different protein precipitation methods systematically, and then the protein was analyzed by SDS-Polyacrylamide Gel Electrophoresis. The recombinant protein was determined with the feature of protein band by the Automated Image Capture and 1-D Analysis Software directly. With this method, the optimized expression conditions of basic fibroblast growth factor secreted from pichia were obtained, which is as the same as using traditional methods. Hence, a convenient tool to determine the optimized conditions for the expression of recombinant proteins in Pichia was established.

  4. An overview on molecular chaperones enhancing solubility of expressed recombinant proteins with correct folding.

    Science.gov (United States)

    Mamipour, Mina; Yousefi, Mohammadreza; Hasanzadeh, Mohammad

    2017-09-01

    The majority of research topics declared that most of the recombinant proteins have been expressed by Escherichia coli in basic investigations. But the majority of high expressed proteins formed as inactive recombinant proteins that are called inclusion body. To overcome this problem, several methods have been used including suitable promoter, environmental factors, ladder tag to secretion of proteins into the periplasm, gene protein optimization, chemical chaperones and molecular chaperones sets. Co-expression of the interest protein with molecular chaperones is one of the common methods The chaperones are a group of proteins, which are involved in making correct folding of recombinant proteins. Chaperones are divided two groups including; cytoplasmic and periplasmic chaperones. Moreover, periplasmic chaperones and proteases can be manipulated to increase the yields of secreted proteins. In this article, we attempted to review cytoplasmic chaperones such as Hsp families and periplasmic chaperones including; generic chaperones, specialized chaperones, PPIases, and proteins involved in disulfide bond formation. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. A high incidence of meiotic silencing of unsynapsed chromatin is not associated with substantial pachytene loss in heterozygous male mice carrying multiple simple robertsonian translocations.

    Directory of Open Access Journals (Sweden)

    Marcia Manterola

    2009-08-01

    Full Text Available Meiosis is a complex type of cell division that involves homologous chromosome pairing, synapsis, recombination, and segregation. When any of these processes is altered, cellular checkpoints arrest meiosis progression and induce cell elimination. Meiotic impairment is particularly frequent in organisms bearing chromosomal translocations. When chromosomal translocations appear in heterozygosis, the chromosomes involved may not correctly complete synapsis, recombination, and/or segregation, thus promoting the activation of checkpoints that lead to the death of the meiocytes. In mammals and other organisms, the unsynapsed chromosomal regions are subject to a process called meiotic silencing of unsynapsed chromatin (MSUC. Different degrees of asynapsis could contribute to disturb the normal loading of MSUC proteins, interfering with autosome and sex chromosome gene expression and triggering a massive pachytene cell death. We report that in mice that are heterozygous for eight multiple simple Robertsonian translocations, most pachytene spermatocytes bear trivalents with unsynapsed regions that incorporate, in a stage-dependent manner, proteins involved in MSUC (e.g., gammaH2AX, ATR, ubiquitinated-H2A, SUMO-1, and XMR. These spermatocytes have a correct MSUC response and are not eliminated during pachytene and most of them proceed into diplotene. However, we found a high incidence of apoptotic spermatocytes at the metaphase stage. These results suggest that in Robertsonian heterozygous mice synapsis defects on most pachytene cells do not trigger a prophase-I checkpoint. Instead, meiotic impairment seems to mainly rely on the action of a checkpoint acting at the metaphase stage. We propose that a low stringency of the pachytene checkpoint could help to increase the chances that spermatocytes with synaptic defects will complete meiotic divisions and differentiate into viable gametes. This scenario, despite a reduction of fertility, allows the spreading

  6. Meiotic Consequences of Genetic Divergence Across the Murine Pseudoautosomal Region

    OpenAIRE

    Dumont, Beth L.

    2017-01-01

    The production of haploid gametes during meiosis is dependent on the homology-driven processes of pairing, synapsis, and recombination. On the mammalian heterogametic sex chromosomes, these key meiotic activities are confined to the pseudoautosomal region (PAR), a short region of near-perfect sequence homology between the X and Y chromosomes. Despite its established importance for meiosis, the PAR is rapidly evolving, raising the question of how proper X/Y segregation is buffered against the ...

  7. Trafficking of endoplasmic reticulum-retained recombinant proteins is unpredictable in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Thomas eDe Meyer

    2014-09-01

    Full Text Available A wide variety of recombinant proteins has been produced in the dicot model plant, Arabidopsis thaliana. Many of these proteins are targeted for secretion by means of an N terminal endoplasmic reticulum (ER signal peptide. In addition, they can also be designed for ER retention by adding a C terminal H/KDEL-tag. Despite extensive knowledge of the protein trafficking pathways, the final protein destination, especially of such H/KDEL-tagged recombinant proteins, is unpredictable. In this respect, glycoproteins are ideal study objects. Microscopy experiments reveal their deposition pattern and characterization of their N-glycans aids in elucidating the trafficking. Here, we combine microscopy and N glycosylation data generated in Arabidopsis leaves and seeds, and highlight the lack of a decent understanding of heterologous protein trafficking.

  8. A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions

    Science.gov (United States)

    Hostetler, Jessica B.; Sharma, Sumana; Bartholdson, S. Josefin; Wright, Gavin J.; Fairhurst, Rick M.; Rayner, Julian C.

    2015-01-01

    suggesting that the proteins are natively folded and functional. This screen also identified two novel protein-protein interactions, between P12 and PVX_110945, and between MSP3.10 and MSP7.1, the latter of which was confirmed by surface plasmon resonance. Conclusions/Significance We produced a new library of recombinant full-length P. vivax ectodomains, established that the majority of them contain tertiary structure, and used them to identify predicted and novel protein-protein interactions. As well as identifying new interactions for further biological studies, this library will be useful in identifying P. vivax proteins with vaccine potential, and studying P. vivax malaria pathogenesis and immunity. Trial Registration ClinicalTrials.gov NCT00663546 PMID:26701602

  9. Bicistronic expression plasmid for the rapid production of recombinant fused proteins in Escherichia coli.

    Science.gov (United States)

    Yero, Daniel; Pajón, Rolando; Niebla, Olivia; Sardiñas, Gretel; Vivar, Isbel; Perera, Yasser; García, Darien; Delgado, Maité; Cobas, Karem

    2006-04-01

    In the post-genomic era, every aspect of the production of proteins must be accelerated. In this way, several vectors are currently exploited for rapid production of recombinant proteins in Escherichia coli. N-terminal fusions to the first 47 amino acids of the LpdA (dihydrolipoamide dehydrogenase A) protein of Neisseria meningitidis have been shown to increase the expression of recombinant proteins. Consequently, we have constructed a modified N-terminal LpdA fusion vector, introducing the blue/white colony selection by exploiting a bicistronic gene organization. In the new vector, the sequence encoding the first 47 amino acids of meningococcal LpdA and the alpha-peptide sequence of beta-galactosidase were connected via a ribosome-binding site, and two MCSs (multiple cloning sites) were located surrounding the latter, allowing efficient cloning by colour selection of recombinants. The vector was also improved with the addition of a C-terminal polyhistidine tag, and an EKS (enterokinase recognition sequence) immediately after the LpdA fusion sequence. The new plasmid was employed in the expression and purification of six different bacterial polypeptides. One of these recombinant proteins, P6 protein from Haemophilus influenzae, was used as a model and its N-terminal fusion sequence was totally removed from the recombinant version after incubation with the enterokinase protease, while the polyhistidine tail successfully allowed the purification of the unfused protein from the protease reaction. Two completely new neisserial vaccine candidates, NMB0088 and NMB1126 proteins, were cloned, expressed and purified using this system. To our knowledge, this constitutes the first report of the cloning and expression of these proteins in E. coli.

  10. RecO protein initiates DNA recombination and strand annealing through two alternative DNA binding mechanisms.

    Science.gov (United States)

    Ryzhikov, Mikhail; Gupta, Richa; Glickman, Michael; Korolev, Sergey

    2014-10-17

    Recombination mediator proteins (RMPs) are important for genome stability in all organisms. Several RMPs support two alternative reactions: initiation of homologous recombination and DNA annealing. We examined mechanisms of RMPs in both reactions with Mycobacterium smegmatis RecO (MsRecO) and demonstrated that MsRecO interacts with ssDNA by two distinct mechanisms. Zinc stimulates MsRecO binding to ssDNA during annealing, whereas the recombination function is zinc-independent and is regulated by interaction with MsRecR. Thus, different structural motifs or conformations of MsRecO are responsible for interaction with ssDNA during annealing and recombination. Neither annealing nor recombinase loading depends on MsRecO interaction with the conserved C-terminal tail of single-stranded (ss) DNA-binding protein (SSB), which is known to bind Escherichia coli RecO. However, similarly to E. coli proteins, MsRecO and MsRecOR do not dismiss SSB from ssDNA, suggesting that RMPs form a complex with SSB-ssDNA even in the absence of binding to the major protein interaction motif. We propose that alternative conformations of such complexes define the mechanism by which RMPs initiate the repair of stalled replication and support two different functions during recombinational repair of DNA breaks. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Application of Recombinant Proteins for Serodiagnosis of Visceral Leishmaniasis in Humans and Dogs.

    Science.gov (United States)

    Farahmand, Mahin; Nahrevanian, Hossein

    2016-07-01

    Visceral leishmaniasis (VL) is a zoonotic disease caused by leishmania species. Dogs are considered to be the main reservoir of VL. A number of methods and antigen-based assays are used for the diagnosis of leishmaniasis. However, currently available methods are mainly based on direct examination of tissues for the presence of parasites, which is highly invasive. A variety of serological tests are commonly applied for VL diagnosis, including indirect fluorescence antibody test, enzyme-linked immunosorbent assay (ELISA), dot-ELISA, direct agglutination test, Western-blotting, and immunochromatographic test. However, when soluble antigens are used, serological tests are less specific due to cross-reactivity with other parasitic diseases. Several studies have attempted to replace soluble antigens with recombinant proteins to improve the sensitivity and the specificity of the immunodiagnostic tests. Major technological advances in recombinant antigens as reagents for the serological diagnosis of VL have led to high sensitivity and specificity of these serological tests. A great number of recombinant proteins have been shown to be effective for the diagnosis of leishmania infection in dogs, the major reservoir of L. infantum. Although few recombinant proteins with high efficacy provide reasonable results for the diagnosis of human and canine VL, more optimization is still needed for the appropriate antigens to provide high-throughput performance. This review aims to explore the application of different recombinant proteins for the serodiagnosis of VL in humans and dogs.

  12. Polymyxin B as inhibitor of LPS contamination of Schistosoma mansoni recombinant proteins in human cytokine analysis

    Directory of Open Access Journals (Sweden)

    Pacífico Lucila G

    2007-01-01

    Full Text Available Abstract Background Recombinant proteins expressed in Escherichia coli vectors are generally contaminated with endotoxin. In this study, we evaluated the ability of Polymyxin B to neutralize the effect of LPS present as contaminant on Schistosoma mansoni recombinant proteins produced in E. coli in inducing TNF-α and IL-10. Peripheral blood mononuclear cells from individuals chronically infected with S. mansoni were stimulated in vitro with recombinant Sm22.6, Sm14 and P24 antigens (10 μg/mL in the presence of Polymyxin B (10 μg/mL. Results The levels of cytokines were measured using ELISA. There was greater than 90 % reduction (p S. mansoni recombinant proteins in the presence of Polymyxin B, a reduction in the levels of TNF-α and IL-10 was also observed. However, the percentage of reduction was lower when compared to the cultures stimulated with LPS, probably because these proteins are able to induce the production of these cytokines by themselves. Conclusion This study showed that Polymyxin B was able to neutralize the effect of endotoxin, as contaminant in S. mansoni recombinant antigens produced in E. coli, in inducing TNF-α and IL-10 production.

  13. Study of recombinant proteins derived from Ser-2 gene of Bombyx mori

    OpenAIRE

    STAŠKOVÁ, Tereza

    2012-01-01

    Four different variants of recombinant proteins derived from Bombyx mori Ser-2 gene were expressed in bacteria. The ability of these proteins to coat hydrofobic surfaces and to support growth of various types of adherent cells in vitro were examined. It was shown that these proteins support cell adhesion and proliferation, and could be used as coating agents to realize surfaces suitable for growth of vertebrate and insect cells.

  14. Production of recombinant proteins in Escherichia coli tagged with the fusion protein CusF3H.

    Science.gov (United States)

    Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Zarate, Xristo

    2017-04-01

    Recombinant protein expression in the bacterium Escherichia coli still is the number one choice for large-scale protein production. Nevertheless, many complications can arise using this microorganism, such as low yields, the formation of inclusion bodies, and the requirement for difficult purification steps. Most of these problems can be solved with the use of fusion proteins. Here, the use of the metal-binding protein CusF3H+ is described as a new fusion protein for recombinant protein expression and purification in E. coli. We have previously shown that CusF produces large amounts of soluble protein, with low levels of formation of inclusion bodies, and that proteins can be purified using IMAC resins charged with Cu(II) ions. CusF3H+ is an enhanced variant of CusF, formed by the addition of three histidine residues at the N-terminus. These residues then can bind Ni(II) ions allowing improved purity after affinity chromatography. Expression and purification of Green Fluorescent Protein tagged with CusF3H+ showed that the mutation did not alter the capacity of the fusion protein to increase protein expression, and purity improved considerably after affinity chromatography with immobilized nickel ions; high yields are obtained after tag-removal since CusF3H+ is a small protein of just 10 kDa. Furthermore, the results of experiments involving expression of tagged proteins having medium to large molecular weights indicate that the presence of the CusF3H+ tag improves protein solubility, as compared to a His-tag. We therefore endorse CusF3H+ as a useful alternative fusion protein/affinity tag for production of recombinant proteins in E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Meiotic Consequences of Genetic Divergence Across the Murine Pseudoautosomal Region.

    Science.gov (United States)

    Dumont, Beth L

    2017-03-01

    The production of haploid gametes during meiosis is dependent on the homology-driven processes of pairing, synapsis, and recombination. On the mammalian heterogametic sex chromosomes, these key meiotic activities are confined to the pseudoautosomal region (PAR), a short region of near-perfect sequence homology between the X and Y chromosomes. Despite its established importance for meiosis, the PAR is rapidly evolving, raising the question of how proper X / Y segregation is buffered against the accumulation of homology-disrupting mutations. Here, I investigate the interplay of PAR evolution and function in two interfertile house mouse subspecies characterized by structurally divergent PARs, Mus musculus domesticus and M. m. castaneus Using cytogenetic methods to visualize the sex chromosomes at meiosis, I show that intersubspecific F 1 hybrids harbor an increased frequency of pachytene spermatocytes with unsynapsed sex chromosomes. This high rate of asynapsis is due, in part, to the premature release of synaptic associations prior to completion of prophase I. Further, I show that when sex chromosomes do synapse in intersubspecific hybrids, recombination is reduced across the paired region. Together, these meiotic defects afflict ∼50% of spermatocytes from F 1 hybrids and lead to increased apoptosis in meiotically dividing cells. Despite flagrant disruption of the meiotic program, a subset of spermatocytes complete meiosis and intersubspecific F 1 males remain fertile. These findings cast light on the meiotic constraints that shape sex chromosome evolution and offer initial clues to resolve the paradox raised by the rapid evolution of this functionally significant locus. Copyright © 2017 by the Genetics Society of America.

  16. Efficient secretory expression of recombinant proteins in Escherichia coli with a novel actinomycete signal peptide.

    Science.gov (United States)

    Cui, Yanbing; Meng, Yiwei; Zhang, Juan; Cheng, Bin; Yin, Huijia; Gao, Chao; Xu, Ping; Yang, Chunyu

    2017-01-01

    In well-established heterologous hosts, such as Escherichia coli, recombinant proteins are usually intracellular and frequently found as inclusion bodies-especially proteins possessing high rare codon content. In this study, successful secretory expression of three hydrolases, in a constructed inducible or constitutive system, was achieved by fusion with a novel signal peptide (Kp-SP) from an actinomycete. The signal peptide efficiently enabled extracellular protein secretion and also contributed to the active expression of the intracellular recombinant proteins. The thermophilic α-amylase gene of Bacillus licheniformis was fused with Kp-SP. Both recombinants, carrying inducible and constitutive plasmids, showed remarkable increases in extracellular and intracellular amylolytic activity. Amylase activity was observed to be > 10-fold in recombinant cultures with the constitutive plasmid, pBSPPc, compared to that in recombinants lacking Kp-SP. Further, the signal peptide enabled efficient secretion of a thermophilic cellulase into the culture medium, as demonstrated by larger halo zones and increased enzymatic activities detected in both constructs from different plasmids. For heterologous proteins with a high proportion of rare codons, it is difficult to obtain high expression in E. coli owing to the codon bias. Here, the fusion of an archaeal homologue of the amylase encoding gene, FSA, with Kp-SP resulted in > 5-fold higher extracellular activity. The successful extracellular expression of the amylase indicated that the signal peptide also contributed significantly to its active expression and signified the potential value of this novel and versatile signal peptide in recombinant protein production. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Enhanced production of recombinant proteins with Corynebacterium glutamicum by deletion of insertion sequences (IS elements).

    Science.gov (United States)

    Choi, Jae Woong; Yim, Sung Sun; Kim, Min Jeong; Jeong, Ki Jun

    2015-12-29

    In most bacteria, various jumping genetic elements including insertion sequences elements (IS elements) cause a variety of genetic rearrangements resulting in harmful effects such as genome and recombinant plasmid instability. The genetic stability of a plasmid in a host is critical for high-level production of recombinant proteins, and in this regard, the development of an IS element-free strain could be a useful strategy for the enhanced production of recombinant proteins. Corynebacterium glutamicum, which is a workhorse in the industrial-scale production of various biomolecules including recombinant proteins, also has several IS elements, and it is necessary to identify the critical IS elements and to develop IS element deleted strain. From the cultivation of C. glutamicum harboring a plasmid for green fluorescent protein (GFP) gene expression, non-fluorescent clones were isolated by FACS (fluorescent activated cell sorting). All the isolated clones had insertions of IS elements in the GFP coding region, and two major IS elements (ISCg1 and ISCg2 families) were identified. By co-cultivating cells harboring either the isolated IS element-inserted plasmid or intact plasmid, it was clearly confirmed that cells harboring the IS element-inserted plasmids became dominant during the cultivation due to their growth advantage over cells containing intact plasmids, which can cause a significant reduction in recombinant protein production during cultivation. To minimize the harmful effects of IS elements on the expression of heterologous genes in C. glutamicum, two IS element free C. glutamicum strains were developed in which each major IS element was deleted, and enhanced productivity in the engineered C. glutamicum strain was successfully demonstrated with three models: GFP, poly(3-hydroxybutyrate) [P(3HB)] and γ-aminobutyrate (GABA). Our findings clearly indicate that the hopping of IS elements could be detrimental to the production of recombinant proteins in C

  18. Serum from Nipah Virus Patients Recognises Recombinant Viral Proteins Produced in Escherichia coli.

    Science.gov (United States)

    Tiong, Vunjia; Lam, Chui-Wan; Phoon, Wai-Hong; AbuBakar, Sazaly; Chang, Li-Yen

    2017-01-24

    The genes for Nipah virus (NiV) proteins were amplified from viral RNA, cloned into the plasmid pTriEx-3 Hygro, expressed, and purified using immobilized metal affinity chromatography. The recombinant N, F, and G NiV proteins (rNiV-N, rNiV-F, and rNiV-G), were successfully expressed in Escherichia coli and purified with a yield of 4, 16, and 4 mg/L, respectively. All 3 recombinant viral proteins reacted with all 19 samples of NiV-positive human sera. The rNiV-N and rNiV-G proteins were the most immunogenic. The recombinant viral proteins did not react with any of the 12 NiV-negative sera. However, serum from a patient with a late-onset relapsing NiV infection complication was found to be primarily reactive to rNiV-G only. Additionally, there is a distinctive variation in the profile of antigen-reactive bands between the sample from a case of relapsing NiV encephalitis and that of acute NiV infection. The overall findings of this study suggest that the recombinant viral proteins have the potential to be developed further for use in the detection of NiV infection, and continuous biosurveillance of NiV infection in resource-limited settings.

  19. N-terminal processing of affinity-tagged recombinant proteins purified by IMAC procedures.

    Science.gov (United States)

    Mooney, Jane T; Fredericks, Dale P; Christensen, Thorkild; Bruun Schiødt, Christine; Hearn, Milton T W

    2015-07-01

    The ability of a new class of metal binding tags to facilitate the purification of recombinant proteins, exemplified by the tagged glutathione S-transferase and human growth hormone, from Escherichia coli fermentation broths and lysates has been further investigated. These histidine-containing tags exhibit high affinity for borderline metal ions chelated to the immobilised ligand, 1,4,7-triazacyclononane (tacn). The use of this tag-tacn immobilised metal ion affinity chromatography (IMAC) system engenders high selectivity with regard to host cell protein removal and permits facile tag removal from the E. coli-expressed recombinant protein. In particular, these tags were specifically designed to enable their efficient removal by the dipeptidyl aminopeptidase 1 (DAP-1), thus capturing the advantages of high substrate specificity and rates of cleavage. MALDI-TOF MS analysis of the cleaved products from the DAP-1 digestion of the recombinant N-terminally tagged proteins confirmed the complete removal of the tag within 4-12 h under mild experimental conditions. Overall, this study demonstrates that the use of tags specifically designed to target tacn-based IMAC resins offers a comprehensive and flexible approach for the purification of E. coli-expressed recombinant proteins, where complete removal of the tag is an essential prerequisite for subsequent application of the purified native proteins in studies aimed at delineating the molecular and cellular basis of specific biological processes. Copyright © 2015 John Wiley & Sons, Ltd.

  20. Analysis of Recombinant Proteins in Transgenic Rice Seeds: Identity, Localization, Tolerance to Digestion, and Plant Stress Response.

    Science.gov (United States)

    Wakasa, Yuhya; Takaiwa, Fumio

    2016-01-01

    Rice seeds are an ideal production platform for high-value recombinant proteins in terms of economy, scalability, safety, and stability. Strategies for the expression of large amounts of recombinant proteins in rice seeds have been established in the past decade and transgenic rice seeds that accumulate recombinant products such as bioactive peptides and proteins, which promote the health and quality of life of humans, have been generated in many laboratories worldwide. One of the most important advantages is the potential for direct oral delivery of transgenic rice seeds without the need for recombinant protein purification (downstream processing), which has been attributed to the high expression levels of recombinant products. Transgenic rice will be beneficial as a delivery system for pharmaceuticals and nutraceuticals in the future. This chapter introduces the strategy for producing recombinant protein in the edible part (endosperm) of the rice grain and describes methods for the analysis of transgenic rice seeds in detail.

  1. Cheese whey-induced high-cell-density production of recombinant proteins in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Neubauer Peter

    2003-04-01

    Full Text Available Abstract Background Use of lactose-rich concentrates from dairy processes for the induction of recombinant gene's expression has not received much attention although they are interesting low cost substrates for production of recombinant enzymes. Applicability of dairy waste for induction of recombinant genes in Escherichia coli was studied. Clones expressing Lactobacillus phage muramidase and Lactobacillus alcohol dehydrogenase were used for the experiments. Results Shake flask cultivations in mineral salt medium showed that cheese whey or deproteinised whey induced gene expression as efficiently as IPTG (isopropyl-β-D-thiogalactopyranoside or pure lactose. Addition of yeast extract or proteolytically degraded whey proteins did not improve the recombinant protein yield. In contrast, addition of yeast extract to the well-balanced mineral salt medium decreased the product yield. Feeding with glycerol provided sufficient amount of easily assimilable carbon source during the induction period without preventing lactose intake and induction by lactose. High-cell-density fed-batch cultivations showed that product yields comparable to IPTG-induction can be achieved by feeding bacteria with a mixture of glycerol and concentrated whey permeate during the induction. Conclusion Whey and concentrated whey permeate can be applied as an alternative inducer in recombinant high-cell-density fed-batch fermentations. The yield of the recombinant product was comparable to fermentations induced by IPTG. In low-cell-density shake flask experiments the yield was higher with whey or whey permeate than with IPTG.

  2. High-resolution structure of the recombinant sweet-tasting protein thaumatin I

    International Nuclear Information System (INIS)

    Masuda, Tetsuya; Ohta, Keisuke; Mikami, Bunzo; Kitabatake, Naofumi

    2011-01-01

    The structure of a recombinant form of the sweet-tasting protein thaumatin I was determined at 1.1 Å resolution and refined to an R work of 9.1% and an R free of 11.7%. Comparisons with plant thaumatin revealed the electron density of recombinant thaumatin I to be significantly improved, especially around Asn46 and Ser63. Thaumatin, an intensely sweet-tasting plant protein, elicits a sweet taste at a concentration of 50 nM. The crystal structure of a recombinant form of thaumatin I produced in the yeast Pichia pastoris has been determined to a resolution of 1.1 Å. The model was refined with anisotropic B parameters and riding H atoms. A comparison of the diffraction data and refinement statistics for recombinant thaumatin I with those for plant thaumatin I revealed no significant differences in the diffraction data. The R values for recombinant thaumatin I and plant thaumatin I (F o > 4σ) were 9.11% and 9.91%, respectively, indicating the final model to be of good quality. Notably, the electron-density maps around Asn46 and Ser63, which differ between thaumatin variants, were significantly improved. Furthermore, a number of H atoms became visible in an OMIT map and could be assigned. The high-quality structure of recombinant thaumatin with H atoms should provide details about sweetness determinants in thaumatin and provide valuable insights into the mechanism of its interaction with taste receptors

  3. Recombinant dioscorins of the yam storage protein expressed in Escherichia coli exhibit antioxidant and immunomodulatory activities.

    Science.gov (United States)

    Jheng, Yi-Jyun; Tsai, Wei-Yi; Chen, Kuo-Hsuan; Lin, Kuo-Wei; Chyan, Chia-Lin; Yang, Ching-Chi; Lin, Kuo-Chih

    2012-09-01

    Dioscorins, the major storage proteins in yam tubers, exhibit biochemical and immunomodulatroy activities. To investigate the potential application of dioscorins in biomedical research, we expressed the dioscorin genes Dj-dioA3 and Dp-dioA2 from Dioscorea japonica and Dioscorea pseudojaponica, respectively, in E. coli and routinely obtained approximately 15 mg proteins per liter Escherichia coli culture (mg/L) to 30 mg/L of rDj-dioscorinA3 and 4 to 8 mg/L of rDp-dioscorinA2. Western blot analyses revealed that both recombinant dioscorins contained epitopes with similar antigenicities to those of the native dioscorins. Results from dithiothreitol (DTT) treatment followed by monobromobimane (mBBr) staining showed that both recombinant dioscorins, like the native dioscorins, contain an intramolecular disulfide bond between Cys(28) and Cys(187) residues. Circular dichroism spectroscopy findings indicated that the secondary structural contents of the recombinant dioscorins showed high similarity to those of their corresponding native dioscorins. Both recombinant dioscorins, like the native dioscorins, exhibited 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and Toll-like receptor 4 signaling activities, and stimulated the phagocytosis of E. coli by macrophage. Overall, our results indicated that substantial amounts of recombinant dioscorins can be purified easily from E. coli and that these recombinant dioscorins are appropriate for application in future investigations of the biomedical functions of dioscorins. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. High-yield secretion of recombinant proteins from the microalga Chlamydomonas reinhardtii.

    Science.gov (United States)

    Ramos-Martinez, Erick Miguel; Fimognari, Lorenzo; Sakuragi, Yumiko

    2017-09-01

    Microalga-based biomanufacturing of recombinant proteins is attracting growing attention due to its advantages in safety, metabolic diversity, scalability and sustainability. Secretion of recombinant proteins can accelerate the use of microalgal platforms by allowing post-translational modifications and easy recovery of products from the culture media. However, currently, the yields of secreted recombinant proteins are low, which hampers the commercial application of this strategy. This study aimed at expanding the genetic tools for enhancing secretion of recombinant proteins in Chlamydomonas reinhardtii, a widely used green microalga as a model organism and a potential industrial biotechnology platform. We demonstrated that the putative signal sequence from C. reinhardtii gametolysin can assist the secretion of the yellow fluorescent protein Venus into the culture media. To increase the secretion yields, Venus was C-terminally fused with synthetic glycomodules comprised of tandem serine (Ser) and proline (Pro) repeats of 10 and 20 units [hereafter (SP) n , wherein n = 10 or 20]. The yields of the (SP) n -fused Venus were higher than Venus without the glycomodule by up to 12-fold, with the maximum yield of 15 mg/L. Moreover, the presence of the glycomodules conferred an enhanced proteolytic protein stability. The Venus-(SP) n proteins were shown to be glycosylated, and a treatment of the cells with brefeldin A led to a suggestion that glycosylation of the (SP) n glycomodules starts in the endoplasmic reticulum (ER). Taken together, the results demonstrate the utility of the gametolysin signal sequence and (SP) n glycomodule to promote a more efficient biomanufacturing of microalgae-based recombinant proteins. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  5. Recombinant Sheep Pox Virus Proteins Elicit Neutralizing Antibodies.

    Science.gov (United States)

    Chervyakova, Olga V; Zaitsev, Valentin L; Iskakov, Bulat K; Tailakova, Elmira T; Strochkov, Vitaliy M; Sultankulova, Kulyaisan T; Sandybayev, Nurlan T; Stanbekova, Gulshan E; Beisenov, Daniyar K; Abduraimov, Yergali O; Mambetaliyev, Muratbay; Sansyzbay, Abylay R; Kovalskaya, Natalia Y; Nemchinov, Lev G; Hammond, Rosemarie W

    2016-06-07

    The aim of this work was to evaluate the immunogenicity and neutralizing activity of sheep pox virus (SPPV; genus Capripoxvirus, family Poxviridae) structural proteins as candidate subunit vaccines to control sheep pox disease. SPPV structural proteins were identified by sequence homology with proteins of vaccinia virus (VACV) strain Copenhagen. Four SPPV proteins (SPPV-ORF 060, SPPV-ORF 095, SPPV-ORF 117, and SPPV-ORF 122), orthologs of immunodominant L1, A4, A27, and A33 VACV proteins, respectively, were produced in Escherichia coli. Western blot analysis revealed the antigenic and immunogenic properties of SPPV-060, SPPV-095, SPPV-117 and SPPV-122 proteins when injected with adjuvant into experimental rabbits. Virus-neutralizing activity against SPPV in lamb kidney cell culture was detected for polyclonal antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. To our knowledge, this is the first report demonstrating the virus-neutralizing activities of antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins.

  6. Recombinant Sheep Pox Virus Proteins Elicit Neutralizing Antibodies

    Directory of Open Access Journals (Sweden)

    Olga V. Chervyakova

    2016-06-01

    Full Text Available The aim of this work was to evaluate the immunogenicity and neutralizing activity of sheep pox virus (SPPV; genus Capripoxvirus, family Poxviridae structural proteins as candidate subunit vaccines to control sheep pox disease. SPPV structural proteins were identified by sequence homology with proteins of vaccinia virus (VACV strain Copenhagen. Four SPPV proteins (SPPV-ORF 060, SPPV-ORF 095, SPPV-ORF 117, and SPPV-ORF 122, orthologs of immunodominant L1, A4, A27, and A33 VACV proteins, respectively, were produced in Escherichia coli. Western blot analysis revealed the antigenic and immunogenic properties of SPPV-060, SPPV-095, SPPV-117 and SPPV-122 proteins when injected with adjuvant into experimental rabbits. Virus-neutralizing activity against SPPV in lamb kidney cell culture was detected for polyclonal antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. To our knowledge, this is the first report demonstrating the virus-neutralizing activities of antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins.

  7. Recombinant fusion protein of albumin-retinol binding protein inactivates stellate cells

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Soyoung; Park, Sangeun; Kim, Suhyun [Laboratory of Cellular Oncology, Korea University Graduate School of Medicine, Ansan, Gyeonggi do 425-707 (Korea, Republic of); Lim, Chaeseung [Department of Laboratory Medicine, Korea University Guro Hospital, Seoul 152-703 (Korea, Republic of); Kim, Jungho [Department of Life Science, Sogang University, Seoul 121-742 (Korea, Republic of); Cha, Dae Ryong [Department of Internal Medicine, Korea University Ansan Hospital, Ansan, Gyeonggi do 425-020 (Korea, Republic of); Oh, Junseo, E-mail: ohjs@korea.ac.kr [Laboratory of Cellular Oncology, Korea University Graduate School of Medicine, Ansan, Gyeonggi do 425-707 (Korea, Republic of)

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer We designed novel recombinant albumin-RBP fusion proteins. Black-Right-Pointing-Pointer Expression of fusion proteins inactivates pancreatic stellate cells (PSCs). Black-Right-Pointing-Pointer Fusion proteins are successfully internalized into and inactivate PSCs. Black-Right-Pointing-Pointer RBP moiety mediates cell specific uptake of fusion protein. -- Abstract: Quiescent pancreatic- (PSCs) and hepatic- (HSCs) stellate cells store vitamin A (retinol) in lipid droplets via retinol binding protein (RBP) receptor and, when activated by profibrogenic stimuli, they transform into myofibroblast-like cells which play a key role in the fibrogenesis. Despite extensive investigations, there is, however, currently no appropriate therapy available for tissue fibrosis. We previously showed that the expression of albumin, composed of three homologous domains (I-III), inhibits stellate cell activation, which requires its high-affinity fatty acid-binding sites asymmetrically distributed in domain I and III. To attain stellate cell-specific uptake, albumin (domain I/III) was coupled to RBP; RBP-albumin{sup domain} {sup III} (R-III) and albumin{sup domain} {sup I}-RBP-albumin{sup III} (I-R-III). To assess the biological activity of fusion proteins, cultured PSCs were used. Like wild type albumin, expression of R-III or I-R-III in PSCs after passage 2 (activated PSCs) induced phenotypic reversal from activated to fat-storing cells. On the other hand, R-III and I-R-III, but not albumin, secreted from transfected 293 cells were successfully internalized into and inactivated PSCs. FPLC-purified R-III was found to be internalized into PSCs via caveolae-mediated endocytosis, and its efficient cellular uptake was also observed in HSCs and podocytes among several cell lines tested. Moreover, tissue distribution of intravenously injected R-III was closely similar to that of RBP. Therefore, our data suggest that albumin-RBP fusion protein comprises

  8. Recombinant Protein Production of Earthworm Lumbrokinase for Potential Antithrombotic Application

    Directory of Open Access Journals (Sweden)

    Kevin Yueju Wang

    2013-01-01

    Full Text Available Earthworms have been used as a traditional medicine in China, Japan, and other Far East countries for thousands of years. Oral administration of dry earthworm powder is considered as a potent and effective supplement for supporting healthy blood circulation. Lumbrokinases are a group of enzymes that were isolated and purified from different species of earthworms. These enzymes are recognized as fibrinolytic agents that can be used to treat various conditions associated with thrombosis. Many lumbrokinase (LK genes have been cloned and characterized. Advances in genetic technology have provided the ability to produce recombinant LK and have made it feasible to purify a single lumbrokinase enzyme for potential antithrombotic application. In this review, we focus on expression systems that can be used for lumbrokinase production. In particular, the advantages of using a transgenic plant system to produce edible lumbrokinase are described.

  9. Development of a lectin binding assay to differentiate between recombinant and endogenous proteins in pharmacokinetic studies of protein-biopharmaceuticals.

    Science.gov (United States)

    Weber, Alfred; Minibeck, Eva; Scheiflinger, Friedrich; Turecek, Peter L

    2015-04-10

    Human glycoproteins, expressed in hamster cell lines, show similar glycosylation patterns to naturally occurring human molecules except for a minute difference in the linkage of terminal sialic acid: both cell types lack α2,6-galactosyl-sialyltransferase, abundantly expressed in human hepatocytes and responsible for the α2,6-sialylation of circulating glycoproteins. This minute difference, which is currently not known to have any physiological relevance, was the basis for the selective measurement of recombinant glycoproteins in the presence of their endogenous counterparts. The assay is based on using the lectin Sambucus nigra agglutinin (SNA), selectively binding to α2,6-sialylated N-glycans. Using von Willebrand factor (VWF), factor IX (FIX), and factor VIIa (FVIIa), it was demonstrated that (i) the plasma-derived proteins, but not the corresponding recombinant proteins, specifically bind to SNA and (ii) this binding can be used to deplete the plasma-derived proteins. The feasibility of this approach was confirmed in spike-recovery studies for all three recombinant coagulation proteins in human plasma and for recombinant VWF (rVWF) in macaque plasma. Analysis of plasma samples from macaques after administration of recombinant and a plasma-derived VWF demonstrated the suitability and robustness of this approach. Data showed that rVWF could be selectively measured without changing the ELISAs and furthermore revealed the limitations of baseline adjustment using a single measurement of the predose concentration only. The SNA gel-based depletion procedure can easily be integrated in existing procedures as a specific sample pre-treatment step. While ELISA-based methods were used to measure the recombinant coagulation proteins in the supernatants obtained by depletion, this procedure is applicable for all biochemical analyses. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Expression and affinity purification of recombinant proteins from plants

    Science.gov (United States)

    Desai, Urvee A.; Sur, Gargi; Daunert, Sylvia; Babbitt, Ruth; Li, Qingshun

    2002-01-01

    With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, beta-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system. Copyright 2002 Elsevier Science (USA).

  11. The Use of Recombinant Hemagglutinine Protein of Rinderpest Virus in Enzyme Immunoassay

    OpenAIRE

    BULUT, Hakan; BOLAT, Yusuf

    2003-01-01

    In this study, Rinderpest virus (RPV) recombinant hemagglutinine protein (rH) fused with protein A region of Staphylococcus aureus was expressed in Escherichia coli and purified by IgG affinity chromatography. rH protein was also used to establish enzyme immunoassay. Therefore, to prevent IgG binding to the protein A the wells coated with the rH proteins were blocked by human serum. Afterwards, RPV antigens were added to the wells to evaluate this assay. To this end, serum from mice immunized...

  12. Rational design of new materials using recombinant structural proteins: Current state and future challenges.

    Science.gov (United States)

    Sutherland, Tara D; Huson, Mickey G; Rapson, Trevor D

    2018-01-01

    Sequence-definable polymers are seen as a prerequisite for design of future materials, with many polymer scientists regarding such polymers as the holy grail of polymer science. Recombinant proteins are sequence-defined polymers. Proteins are dictated by DNA templates and therefore the sequence of amino acids in a protein is defined, and molecular biology provides tools that allow redesign of the DNA as required. Despite this advantage, proteins are underrepresented in materials science. In this publication we investigate the advantages and limitations of using proteins as templates for rational design of new materials. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

  13. Suppression of genetic recombination in the pseudoautosomal region and at subtelomeres in mice with a hypomorphic Spo11 allele.

    Science.gov (United States)

    Smagulova, Fatima; Brick, Kevin; Pu, Yongmei; Sengupta, Uttara; Camerini-Otero, R Daniel; Petukhova, Galina V

    2013-07-22

    Homologous recombination is the key process that generates genetic diversity and drives evolution. SPO11 protein triggers recombination by introducing DNA double stranded breaks at discreet areas of the genome called recombination hotspots. The hotspot locations are largely determined by the DNA binding specificity of the PRDM9 protein in human, mice and most other mammals. In budding yeast Saccharomyces cerevisae, which lacks a Prdm9 gene, meiotic breaks are formed opportunistically in the regions of accessible chromatin, primarily at gene promoters. The genome-wide distribution of hotspots in this organism can be altered by tethering Spo11 protein to Gal4 recognition sequences in the strain expressing Spo11 attached to the DNA binding domain of the Gal4 transcription factor. To establish whether similar re-targeting of meiotic breaks can be achieved in PRDM9-containing organisms we have generated a Gal4BD-Spo11 mouse that expresses SPO11 protein joined to the DNA binding domain of yeast Gal4. We have mapped the genome-wide distribution of the recombination initiation sites in the Gal4BD-Spo11 mice. More than two hundred of the hotspots in these mice were novel and were likely defined by Gal4BD, as the Gal4 consensus motif was clustered around the centers in these hotspots. Surprisingly, meiotic DNA breaks in the Gal4BD-Spo11 mice were significantly depleted near the ends of chromosomes. The effect is particularly striking at the pseudoautosomal region of the X and Y chromosomes - normally the hottest region in the genome. Our data suggest that specific, yet-unidentified factors influence the initiation of meiotic recombination at subtelomeric chromosomal regions.

  14. Smart sustainable bottle (SSB) system for E. coli based recombinant protein production.

    Science.gov (United States)

    Li, Zhaopeng; Carstensen, Bettina; Rinas, Ursula

    2014-11-05

    Recombinant proteins are usually required in laboratories interested in the protein but not in the production process itself. Thus, technical equipment which is easy to handle and straight forward protein production procedures are of great benefit to those laboratories. Companies selling single use cultivation bags and bioreactors are trying to satisfy at least part of these needs. However, single-use systems can contribute to major costs which might be acceptable when "good manufacturing practices" are required but not acceptable for most laboratories facing tight funding. The assembly and application of a simple self-made "smart sustainable bottle" (SSB) system for E. coli based protein production is presented. The core of the SSB system is a 2-L glass bottle which is operated at constant temperature, air flow, and stirrer speed without measurement and control of pH and dissolved oxygen. Oxygen transfer capacities are in the range as in conventional bioreactors operated at intermediate aeration rates and by far exceed those found in conventional shaking flasks and disposable bioreactors. The SSB system was applied for the production of various recombinant proteins using T7-based expression systems and a defined autoinduction medium. The production performance regarding amount and solubility of proteins with robust and delicate properties was as good as in state-of-the-art stirred tank commercial bioreactors. The SSB system represents a low cost protein production device applicable for easy, effective, and reproducible recombinant protein production.

  15. Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins.

    Science.gov (United States)

    Mamat, Uwe; Wilke, Kathleen; Bramhill, David; Schromm, Andra Beate; Lindner, Buko; Kohl, Thomas Andreas; Corchero, José Luis; Villaverde, Antonio; Schaffer, Lana; Head, Steven Robert; Souvignier, Chad; Meredith, Timothy Charles; Woodard, Ronald Wesley

    2015-04-16

    Lipopolysaccharide (LPS), also referred to as endotoxin, is the major constituent of the outer leaflet of the outer membrane of virtually all Gram-negative bacteria. The lipid A moiety, which anchors the LPS molecule to the outer membrane, acts as a potent agonist for Toll-like receptor 4/myeloid differentiation factor 2-mediated pro-inflammatory activity in mammals and, thus, represents the endotoxic principle of LPS. Recombinant proteins, commonly manufactured in Escherichia coli, are generally contaminated with endotoxin. Removal of bacterial endotoxin from recombinant therapeutic proteins is a challenging and expensive process that has been necessary to ensure the safety of the final product. As an alternative strategy for common endotoxin removal methods, we have developed a series of E. coli strains that are able to grow and express recombinant proteins with the endotoxin precursor lipid IVA as the only LPS-related molecule in their outer membranes. Lipid IVA does not trigger an endotoxic response in humans typical of bacterial LPS chemotypes. Hence the engineered cells themselves, and the purified proteins expressed within these cells display extremely low endotoxin levels. This paper describes the preparation and characterization of endotoxin-free E. coli strains, and demonstrates the direct production of recombinant proteins with negligible endotoxin contamination.

  16. The quaternary structure of the recombinant bovine odorant-binding protein is modulated by chemical denaturants.

    Directory of Open Access Journals (Sweden)

    Olga V Stepanenko

    Full Text Available A large group of odorant-binding proteins (OBPs has attracted great scientific interest as promising building blocks in constructing optical biosensors for dangerous substances, such as toxic and explosive molecules. Native tissue-extracted bovine OBP (bOBP has a unique dimer folding pattern that involves crossing the α-helical domain in each monomer over the other monomer's β-barrel. In contrast, recombinant bOBP maintaining the high level of stability inherent to native tissue bOBP is produced in a stable native-like state with a decreased tendency for dimerization and is a mixture of monomers and dimers in a buffered solution. This work is focused on the study of the quaternary structure and the folding-unfolding processes of the recombinant bOBP in the absence and in the presence of guanidine hydrochloride (GdnHCl. Our results show that the recombinant bOBP native dimer is only formed at elevated GdnHCl concentrations (1.5 M. This process requires re-organizing the protein structure by progressing through the formation of an intermediate state. The bOBP dimerization process appears to be irreversible and it occurs before the protein unfolds. Though the observed structural changes for recombinant bOBP at pre-denaturing GdnHCl concentrations show a local character and the overall protein structure is maintained, such changes should be considered where the protein is used as a sensitive element in a biosensor system.

  17. The effect of alcohol on recombinant proteins derived from mammalian adenylyl cyclase

    Directory of Open Access Journals (Sweden)

    Emily Qualls-Creekmore

    2017-07-01

    Full Text Available The cyclic AMP (cAMP signaling pathway is implicated in the development of alcohol use disorder. Previous studies have demonstrated that ethanol enhances the activity of adenylyl cyclase (AC in an isoform specific manner; AC7 is most enhanced by ethanol, and regions responsible for enhancement by ethanol are located in the cytoplasmic domains of the AC7 protein. We hypothesize that ethanol modulates AC activity by directly interacting with the protein and that ethanol effects on AC can be studied using recombinant AC in vitro. AC recombinant proteins containing only the C1a or C2 domains of AC7 and AC9 individually were expressed in bacteria, and purified. The purified recombinant AC proteins retained enzymatic activity and isoform specific alcohol responsiveness. The combination of the C1a or C2 domains of AC7 maintained the same alcohol cutoff point as full-length AC7. We also find that the recombinant AC7 responds to alcohol differently in the presence of different combinations of activators including MnCl2, forskolin, and Gsα. Through a series of concentration-response experiments and curve fitting, the values for maximum activities, Hill coefficients, and EC50 were determined in the absence and presence of butanol as a surrogate of ethanol. The results suggest that alcohol modulates AC activity by directly interacting with the AC protein and that the alcohol interaction with the AC protein occurs at multiple sites with positive cooperativity. This study indicates that the recombinant AC proteins expressed in bacteria can provide a useful model system to investigate the mechanism of alcohol action on their activity.

  18. that Bind Specifically to Recombinant Envelope Protein of Dengue

    African Journals Online (AJOL)

    Tropical Journal of Pharmaceutical Research June 2015; 14 (6): 997-1003 ... Revised accepted: 30 April 2015. Abstract ... Results: The 45 KDa, 43 KDa and 30 KDa plasma membrane proteins were identified as viral envelope targets.

  19. Origin of meiotic nondisjunction in Drosophila females

    International Nuclear Information System (INIS)

    Grell, R.F.

    1978-01-01

    Meiotic nondisjunction can be induced by external agents, such as heat, radiation, and chemicals, and by internal genotypic alterations, namely, point mutations and chromosomal rearrangements. In many cases nondisjunction arises from a reduction or elimination of crossing-over, leading to the production of homologous univalents which fail to co-orient on the metaphase plate and to disjoin properly. In some organisms, e.g., Drosophila and perhaps man, distributive pairing [i.e., a post-exchange, size-dependent pairing] ensures the regular segregation of such homologous univalents. When a nonhomologous univalent is present, which falls within a size range permitting nonhomologous recognition and pairing, distributive nondisjunction of the homologues may follow. Examples of nondisjunction induced by inversion heterozygosity, translocation heterozygosity, chromosome fragments, radiation, heat, and recombination-defective mutants are presented

  20. Obtaining classical swine fever virus E2 recombinant protein and DNA-vaccine on the basis of one subunit

    International Nuclear Information System (INIS)

    Deryabin, O.; Deryabina, O.; Verbitskiy, P.; Kordyum, V.

    2005-01-01

    Three forms of E2 recombinant protein were expressed in E. coli. Swine sera obtained against different forms of the recombinant protein were cross-studied with indirect ELISA. Using individual proteins as an antigen, only 15% of sera against other forms of protein reacted positively, while 100% of heterologous sera showed positive reaction with fused protein. Challenge experiments showed the existence of protective action only from the individual protein. Specificity and activity of sera obtained from the animals after control challenge was confirmed in a blocking variant of ELISA. Genetic construction used a eukaryotic vector that contained the E2 protein gene. Immunization of mice with the resulting DNA induced synthesis of specific antibodies, the titre of which increased considerably after additional single immunization with the E2 recombinant protein, expressed in E. coli. This demonstrated the effectiveness of animal priming by DNA vaccine, and the possibility of using the E2 recombinant protein in E. coli for booster vaccination. (author)

  1. Meiotic double-strand breaks at the interface of chromosome movement, chromosome remodeling, and reductional division

    Science.gov (United States)

    Storlazzi, Aurora; Tessé, Sophie; Gargano, Silvana; James, Françoise; Kleckner, Nancy; Zickler, Denise

    2003-01-01

    Chromosomal processes related to formation and function of meiotic chiasmata have been analyzed in Sordaria macrospora. Double-strand breaks (DSBs), programmed or γ-rays-induced, are found to promote four major events beyond recombination and accompanying synaptonemal complex formation: (1) juxtaposition of homologs from long-distance interactions to close presynaptic coalignment at midleptotene; (2) structural destabilization of chromosomes at leptotene/zygotene, including sister axis separation and fracturing, as revealed in a mutant altered in the conserved, axis-associated cohesin-related protein Spo76/Pds5p; (3) exit from the bouquet stage, with accompanying global chromosome movements, at zygotene/pachytene (bouquet stage exit is further found to be a cell-wide regulatory transition and DSB transesterase Spo11p is suggested to have a new noncatalytic role in this transition); (4) normal occurrence of both meiotic divisions, including normal sister separation. Functional interactions between DSBs and the spo76-1 mutation suggest that Spo76/Pds5p opposes local destabilization of axes at developing chiasma sites and raise the possibility of a regulatory mechanism that directly monitors the presence of chiasmata at metaphase I. Local chromosome remodeling at DSB sites appears to trigger an entire cascade of chromosome movements, morphogenetic changes, and regulatory effects that are superimposed upon a foundation of DSB-independent processes. PMID:14563680

  2. Respiratory syncytial virus subunit vaccine based on a recombinant fusion protein expressed transiently in mammalian cells.

    Science.gov (United States)

    Nallet, Sophie; Amacker, Mario; Westerfeld, Nicole; Baldi, Lucia; König, Iwo; Hacker, David L; Zaborosch, Christiane; Zurbriggen, Rinaldo; Wurm, Florian M

    2009-10-30

    Although respiratory syncytial virus (RSV) causes severe lower respiratory tract infection in infants and adults at risk, no RSV vaccine is currently available. In this report, efforts toward the generation of an RSV subunit vaccine using recombinant RSV fusion protein (rRSV-F) are described. The recombinant protein was produced by transient gene expression (TGE) in suspension-adapted human embryonic kidney cells (HEK-293E) in 4 L orbitally shaken bioreactors. It was then purified and formulated in immunostimulating reconstituted influenza virosomes (IRIVs). The candidate vaccine induced anti-RSV-F neutralizing antibodies in mice, and challenge studies in cotton rats are ongoing. If successful in preclinical and clinical trials, this will be the first recombinant subunit vaccine produced by large-scale TGE in mammalian cells.

  3. [Expression and activity determination of recombinant capsid protein VP2 gene of enterovirus type 71].

    Science.gov (United States)

    Huang, Xueyong; Liu, Guohua; Hu, Xiaoning; Du, Yanhua; Li, Xingle; Xu, Yuling; Chen, Haomin; Xu, Bianli

    2014-04-01

    To clone and express the recombinant capsid protein VP2 of enterovirus type 71 (EV71) and to identify the immune activity of expressed protein in order to build a basis for the investigation work of vaccine and diagnostic antigen. VP2 gene of EV71 was amplified by PCR, and then was cut by restriction enzyme and inserted into expression vector pMAL-c2X. The positive recombinants were transferred into E.coli TB1, the genetically engineered bacteria including pMAL-c2X-VP2 plasmids were induced by isopropyl thiogalactoside ( IPTG) , and the expression products were analyzed by SDS-PAGE and western blotting method. EV71 IgM antibody detection method by ELISA was set up, and the sensitivity and specificity of this method was assessed; 60 neutralizing antibody positive serum samples from hand foot and mouth disease (HFMD) patients were determined, of which 52 samples were positive and 8 samples were negative; a total of 88 acute phase serum samples of HFMD patients diagnosed in clinical were also detected. VP2 gene of 762 bp was obtained by PCR, the gene segment inserted into the recombinant vector was identified using restriction enzyme digestion. The recombinant vector could express a specific about 71 500 fusion protein in E.coli by SDS-PAGE. The purified recombinant protein of EV71-VP2 can react with the serum of HFMD patients to produce a specific band by western blotting. The sensitivity and specificity of ELISA was 87% and 83%, respectively. Of the 88 acute phase serum samples from children with HFMD, 48 samples (55%) were positive by the ELISA assay. VP2 gene of EV71 has been cloned and a prokaryotic high expression system for VP2 gene was successfully constructed in the present study. The recombination EV71-VP2 has well antigenicity, which could be useful for developing diagnose reagent or vaccine of EV71.

  4. A novel genetic system for recombinant protein secretion in the Antarctic Pseudoalteromonas haloplanktis TAC125

    Directory of Open Access Journals (Sweden)

    Marino Gennaro

    2006-12-01

    Full Text Available Abstract Background The final aim of recombinant protein production is both to have a high specific production rate and a high product quality. It was already shown that using cold-adapted bacteria as host vectors, some "intractable" proteins can be efficiently produced at temperature as low as 4°C. Results A novel genetic system for the production and secretion of recombinant proteins in the Antarctic Gram-negative bacterium Pseudoalteromonas haloplanktis TAC125 was set up. This system aims at combining the low temperature recombinant product production with the advantages of extra-cellular protein targeting. The psychrophilic α-amylase from Pseudoalteromonas haloplanktis TAB23 was used as secretion carrier. Three chimerical proteins were produced by fusing intra-cellular proteins to C-terminus of the psychrophilic α-amylase and their secretion was analysed. Data reported in this paper demonstrate that all tested chimeras were translocated with a secretion yield always higher than 80%. Conclusion Data presented here demonstrate that the "cold" gene-expression system is efficient since the secretion yield of tested chimeras is always above 80%. These secretion performances place the α-amylase derived secretion system amongst the best heterologous secretion systems in Gram-negative bacteria reported so far. As for the quality of the secreted passenger proteins, data presented suggest that the system also allows the correct disulphide bond formation of chimera components, secreting a fully active passenger.

  5. A dual protease approach for expression and affinity purification of recombinant proteins.

    Science.gov (United States)

    Raran-Kurussi, Sreejith; Waugh, David S

    2016-07-01

    We describe a new method for affinity purification of recombinant proteins using a dual protease protocol. Escherichia coli maltose binding protein (MBP) is employed as an N-terminal tag to increase the yield and solubility of its fusion partners. The MBP moiety is then removed by rhinovirus 3C protease, prior to purification, to yield an N-terminally His6-tagged protein. Proteins that are only temporarily rendered soluble by fusing them to MBP are readily identified at this stage because they will precipitate after the MBP tag is removed by 3C protease. The remaining soluble His6-tagged protein, if any, is subsequently purified by immobilized metal affinity chromatography (IMAC). Finally, the N-terminal His6 tag is removed by His6-tagged tobacco etch virus (TEV) protease to yield the native recombinant protein, and the His6-tagged contaminants are removed by adsorption during a second round of IMAC, leaving only the untagged recombinant protein in the column effluent. The generic strategy described here saves time and effort by removing insoluble aggregates at an early stage in the process while also reducing the tendency of MBP to "stick" to its fusion partners during affinity purification. Published by Elsevier Inc.

  6. Roles of silkworm endoplasmic reticulum chaperones in the secretion of recombinant proteins expressed by baculovirus system.

    Science.gov (United States)

    Imai, Saki; Kusakabe, Takahiro; Xu, Jian; Li, Zhiqing; Shirai, Shintaro; Mon, Hiroaki; Morokuma, Daisuke; Lee, Jae Man

    2015-11-01

    Baculovirus expression vector system (BEVS) is widely used for production of recombinant eukaryotic proteins in insect larvae or cultured cells. BEVS has advantages over bacterial expression system in producing post-translationally modified secreted proteins. However, for some unknown reason, it is very difficult for insects to secrete sufficiently for certain proteins of interest. To understand the reasons why insect cells fail to secrete some kinds of recombinant proteins, we here employed three mammalian proteins as targets, EPO, HGF, and Wnt3A, with different secretion levels in BEVS and investigated their mRNA transcriptions from the viral genome, subcellular localizations, and interactions with silkworm ER chaperones. Moreover, we observed that no significantly influence on the secretion amounts of all three proteins when depleting or overexpressing most endogenous ER chaperone genes in cultured silkworm cells. However, among all detected ER chaperones, the depletion of BiP severely decreased the recombinant protein secretion in BEVS, indicating the possible central role of Bip in silkworm secretion pathway.

  7. Genome-scale metabolic model of Pichia pastoris with native and humanized glycosylation of recombinant proteins.

    Science.gov (United States)

    Irani, Zahra Azimzadeh; Kerkhoven, Eduard J; Shojaosadati, Seyed Abbas; Nielsen, Jens

    2016-05-01

    Pichia pastoris is used for commercial production of human therapeutic proteins, and genome-scale models of P. pastoris metabolism have been generated in the past to study the metabolism and associated protein production by this yeast. A major challenge with clinical usage of recombinant proteins produced by P. pastoris is the difference in N-glycosylation of proteins produced by humans and this yeast. However, through metabolic engineering, a P. pastoris strain capable of producing humanized N-glycosylated proteins was constructed. The current genome-scale models of P. pastoris do not address native nor humanized N-glycosylation, and we therefore developed ihGlycopastoris, an extension to the iLC915 model with both native and humanized N-glycosylation for recombinant protein production, but also an estimation of N-glycosylation of P. pastoris native proteins. This new model gives a better prediction of protein yield, demonstrates the effect of the different types of N-glycosylation of protein yield, and can be used to predict potential targets for strain improvement. The model represents a step towards a more complete description of protein production in P. pastoris, which is required for using these models to understand and optimize protein production processes. © 2015 Wiley Periodicals, Inc.

  8. Refolding Techniques for Recovering Biologically Active Recombinant Proteins from Inclusion Bodies

    Directory of Open Access Journals (Sweden)

    Hiroshi Yamaguchi

    2014-02-01

    Full Text Available Biologically active proteins are useful for studying the biological functions of genes and for the development of therapeutic drugs and biomaterials in a biotechnology industry. Overexpression of recombinant proteins in bacteria, such as Escherichia coli, often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. As inclusion bodies contain relatively pure and intact proteins, protein refolding is an important process to obtain active recombinant proteins from inclusion bodies. However, conventional refolding methods, such as dialysis and dilution, are time consuming and, often, recovered yields of active proteins are low, and a trial-and-error process is required to achieve success. Recently, several approaches have been reported to refold these aggregated proteins into an active form. The strategies largely aim at reducing protein aggregation during the refolding procedure. This review focuses on protein refolding techniques using chemical additives and laminar flow in microfluidic chips for the efficient recovery of active proteins from inclusion bodies.

  9. Chemical Ligation of Folded Recombinant Proteins: Segmental Isotopic Labeling of Domains for NMR Studies

    Science.gov (United States)

    Xu, Rong; Ayers, Brenda; Cowburn, David; Muir, Tom W.

    1999-01-01

    A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homology type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory apparatus of the protein, were individually prepared in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prepare NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with 15N Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prepare recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chemical ligation in vitro, will increase the size limits for protein structural determination in solution with NMR methods. In vitro chemical ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains.

  10. Analysis of the lipidated recombinant outer surface protein A from Borrelia burgdorferi by mass spectrometry

    NARCIS (Netherlands)

    Bouchon, B.; Klein, Michele; Bischoff, Rainer; Van Dorsselaer, A.; Roitsch, C.

    1997-01-01

    The outer surface protein A, OspA, from the spirochete Borrelia burgdorferi is a lipoprotein of 25 kDa. The recombinant OspA (rOspA) expressed in Escherichia coli has been purified and analyzed by electrospray mass spectrometry (ESMS). A heterogenous spectrum gave a measured mass of 28,462 +/- 9 Da

  11. IgM-specific serodiagnosis of acute human cytomegalovirus infection using recombinant autologous fusion proteins

    NARCIS (Netherlands)

    Vornhagen, R; Hinderer, W; Sonneborn, HH; Bein, G; Matter, L; The, T. Hauw; Enders, G; Jahn, G; Plachter, B

    Portions of three human cytomegalovirus (HCMV) polypeptides, which were shown previously to be highly reactive with patient sera, were expressed in Escherichia coli as autologous fusion proteins. Purified recombinant polypeptides were used as antigens in enzyme linked immunosorbent assay (ELISA) and

  12. Human recombinant protein C for severe sepsis and septic shock in adult and paediatric patients

    DEFF Research Database (Denmark)

    Martí-Carvajal, Arturo J; Solà, Ivan; Gluud, Christian

    2012-01-01

    Sepsis is a common and frequently fatal condition. Human recombinant activated protein C (APC) has been introduced to reduce the high risk of death associated with severe sepsis or septic shock. This systematic review is an update of a Cochrane review originally published in 2007....

  13. Production of recombinant proteins GST L1, E6 and E7 tag HPV 16 ...

    African Journals Online (AJOL)

    In the present work recombinant proteins were produced for used in LUMINEX in order to undergo serological study of Tunisian female population. HPV types 16 L1, E6 and E7 sequences fused to their 3'-end to a sequence encoding the terminal undecapeptide of the SV40 large T-antigen (tag) were isolated from plasmids ...

  14. Production of Polyclonal Antibodies to a Recombinant Coat Protein of Potato mop-top virus

    Czech Academy of Sciences Publication Activity Database

    Čeřovská, Noemi; Moravec, Tomáš; Rosecká, Pavla; Dědič, P.; Filigarová, Marie

    2003-01-01

    Roč. 151, č. 4 (2003), s. 195-200 ISSN 0931-1785 R&D Projects: GA ČR GA522/01/1121 Institutional research plan: CEZ:AV0Z5038910 Keywords : potato mop-top virus * recombinant coat protein * Escherichia Coli Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.557, year: 2003

  15. Polyclonal Antibodies to a Recombinant Coat Protein of Potato Virus A

    Czech Academy of Sciences Publication Activity Database

    Čeřovská, Noemi; Moravec, Tomáš; Velemínský, Jiří

    2002-01-01

    Roč. 46, - (2002), s. 147-151 ISSN 0001-723X R&D Projects: GA ČR GA310/00/0381 Institutional research plan: CEZ:AV0Z5038910 Keywords : Potato virus A * recombinant coat protein * Escherichia coli Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.660, year: 2002

  16. A novel multi-epitope recombined protein for diagnosis of human brucellosis.

    Science.gov (United States)

    Yin, Dehui; Li, Li; Song, Xiuling; Li, Han; Wang, Juan; Ju, Wen; Qu, Xiaofeng; Song, Dandan; Liu, Yushen; Meng, Xiangjun; Cao, Hongqian; Song, Weiyi; Meng, Rizeng; Liu, Jinhua; Li, Juan; Xu, Kun

    2016-05-21

    In epidemic regions of the world, brucellosis is a reemerging zoonosis with minimal mortality but is a serious public hygiene problem. Currently, there are various methods for brucellosis diagnosis, however few of them are available to be used to diagnose, especially for serious cross-reaction with other bacteria. To overcome this disadvantage, we explored a novel multi-epitope recombinant protein as human brucellosis diagnostic antigen. We established an indirect enzyme-linked immunosorbent assay (ELISA) based on this recombinant protein. 248 sera obtained from three different groups including patients with brucellosis (146 samples), non-brucellosis patients (82 samples), and healthy individuals (20 samples) were tested by indirect ELISA. To evaluate the assay, a receiver-operating characteristic (ROC) analysis and immunoblotting were carried out using these characterized serum samples. For this test, the area under the ROC curve was 0.9409 (95 % confidence interval, 0.9108 to 0.9709), and a sensitivity of 88.89 % and a specificity of 85.54 % was given with a cutoff value of 0.3865 from this ROC analysis. The Western blot results indicate that it is feasible to differentiate human brucellosis and non-brucellosis with the newly established method based on this recombinant protein. Our results obtained high diagnostic accuracy of the ELISA assay which encourage the use of this novel recombinant protein as diagnostic antigen to implement serological diagnosis of brucellosis.

  17. Mre11 and Exo1 contribute to the initiation and processivity of resection at meiotic double-strand breaks made independently of Spo11.

    Science.gov (United States)

    Hodgson, Adam; Terentyev, Yaroslav; Johnson, Rebecca A; Bishop-Bailey, Anna; Angevin, Thibaut; Croucher, Adam; Goldman, Alastair S H

    2011-02-07

    During meiosis DNA double-strand breaks (DSBs) are induced and repaired by homologous recombination to create gene conversion and crossover products. Mostly these DSBs are made by Spo11, which covalently binds to the DSB ends. More rarely in Saccharomyces cerevisiae, other meiotic DSBs are formed by self-homing endonucleases such as VDE, which is site specific and does not covalently bind to the DSB ends. We have used experimentally located VDE-DSB sites to analyse an intermediate step in homologous recombination, resection of the single-strand ending 5' at the DSB site. Analysis of strains with different mutant alleles of MRE11 (mre11-58S and mre11-H125N) and deleted for EXO1 indicated that these two nucleases make significant contributions to repair of VDE-DSBs. Physical analysis of single-stranded repair intermediates indicates that efficient initiation and processivity of resection at VDE-DSBs require both Mre11 and Exo1, with loss of function for either protein causing severe delay in resection. We propose that these experiments model what happens at Spo11-DSBs after removal of the covalently bound protein, and that Mre11 and Exo1 are the major nucleases involved in creating resection tracts of widely varying lengths typical of meiotic recombination. Copyright © 2010 Elsevier B.V. All rights reserved.

  18. Molecular chaperone assisted expression systems: obtaining pure soluble and active recombinant proteins for structural and therapeutic purposes

    CSIR Research Space (South Africa)

    Makhoba, XH

    2015-09-01

    Full Text Available For many years recombinant protein production has been at the center of biosciences used for structural and therapeutic purposes. The production of recombinant proteins in foreign host system such as E. coli has been a biggest challenge. This has...

  19. Mechanisms and Regulation of Mitotic Recombination in Saccharomyces cerevisiae

    Science.gov (United States)

    Symington, Lorraine S.; Rothstein, Rodney; Lisby, Michael

    2014-01-01

    Homology-dependent exchange of genetic information between DNA molecules has a profound impact on the maintenance of genome integrity by facilitating error-free DNA repair, replication, and chromosome segregation during cell division as well as programmed cell developmental events. This chapter will focus on homologous mitotic recombination in budding yeast Saccharomyces cerevisiae. However, there is an important link between mitotic and meiotic recombination (covered in the forthcoming chapter by Hunter et al. 2015) and many of the functions are evolutionarily conserved. Here we will discuss several models that have been proposed to explain the mechanism of mitotic recombination, the genes and proteins involved in various pathways, the genetic and physical assays used to discover and study these genes, and the roles of many of these proteins inside the cell. PMID:25381364

  20. Protection by recombinant viral proteins against a respiratory challenge with virulent avian metapneumovirus.

    Science.gov (United States)

    Chary, Parag; Njenga, M Kariuki; Sharma, Jagdev M

    2005-12-15

    Protection by recombinant avian metapneumovirus (aMPV) N or M proteins against a respiratory challenge with virulent aMPV was examined. N, M or N+M proteins were administered intramuscularly (IM) with incomplete Freund's adjuvant (IFA) or by the oculonasal (ON) route with cholera toxin-B (CTB). Each turkey received 40 or 80 microg of each recombinant protein. Birds were considered protected against challenge if the challenge virus was not detectable in the choanal swabs by RT-PCR. At a dose of 40 microg/bird, N protein given with IFA by the IM route protected eight out of nine birds. M protein at the same dose protected three out of seven birds, while a combination of N+M proteins (40 microg each) protected three out of four birds. At a dose of 80 microg of each of N and M proteins per bird given with IFA by the IM route, 100% protection was achieved. ON immunization with a mixture of N and M proteins induced partial protection when the proteins were given with CTB; no detectable protection was noted without CTB. N and M proteins induced anti-aMPV antibodies, although protection against virulent virus challenge did not appear to be associated with the level or presence of antibodies.

  1. New sensitive and specific assay for human immunodeficiency virus antibodies using labeled recombinant fusion protein and time-resolved fluoroimmunoassay.

    OpenAIRE

    Siitari, H; Turunen, P; Schrimsher, J; Nunn, M

    1990-01-01

    A new, rapid method for the detection of human immunodeficiency virus type 1 (HIV-1) antibody by time-resolved fluoroimmunoassay (TR-FIA) was developed. In this assay format, microtitration strips were coated with a recombinant fusion protein, and the same protein was labeled with europium and added into the wells simultaneously with the test specimens. The recombinant fusion protein contained the HIV-1 p24 gag protein sequence that carried an insertion, near the carboxyl terminus, of a 23-am...

  2. Immunization against Rumen Methanogenesis by Vaccination with a New Recombinant Protein.

    Directory of Open Access Journals (Sweden)

    Litai Zhang

    Full Text Available Vaccination through recombinant proteins against rumen methanogenesis provides a mitigation approach to reduce enteric methane (CH4 emissions in ruminants. The objective of present study was to evaluate the in vivo efficacy of a new vaccine candidate protein (EhaF on methanogenesis and microbial population in the rumen of goats. We amplified the gene mru 1407 encoding protein EhaF using fresh rumen fluid samples of mature goats and successfully expressed recombinant protein (EhaF in Escherichia coli Rosetta. This product was evaluated using 12 mature goats with half for control and other half injected with 400ug/goat the purified recombinant protein in day 1 and two subsequent booster immunizations in day 35 and 49. All measurements were undertaken from 63 to 68 days after the initial vaccination, with CH4 emissions determined using respiration calorimeter chambers. The results showed that the vaccination caused intensive immune responses in serum and saliva, although it had no significant effect on total enteric CH4 emissions and methanogen population in the rumen, when compared with the control goats. However, the vaccination altered the composition of rumen bacteria, especially the abundance of main phylum Firmicutes and genus Prevotella. The results indicate that protein EhaF might not be an effective vaccine to reduce enteric CH4 emissions but our vaccine have potential to influence the rumen ecosystem of goats.

  3. Advanced technologies for improved expression of recombinant proteins in bacteria: perspectives and applications.

    Science.gov (United States)

    Gupta, Sanjeev K; Shukla, Pratyoosh

    2016-12-01

    Prokaryotic expression systems are superior in producing valuable recombinant proteins, enzymes and therapeutic products. Conventional microbial technology is evolving gradually and amalgamated with advanced technologies in order to give rise to improved processes for the production of metabolites, recombinant biopharmaceuticals and industrial enzymes. Recently, several novel approaches have been employed in a bacterial expression platform to improve recombinant protein expression. These approaches involve metabolic engineering, use of strong promoters, novel vector elements such as inducers and enhancers, protein tags, secretion signals, high-throughput devices for cloning and process screening as well as fermentation technologies. Advancement of the novel technologies in E. coli systems led to the production of "difficult to express" complex products including small peptides, antibody fragments, few proteins and full-length aglycosylated monoclonal antibodies in considerable large quantity. Wacker's secretion technologies, Pfenex system, inducers, cell-free systems, strain engineering for post-translational modification, such as disulfide bridging and bacterial N-glycosylation, are still under evaluation for the production of complex proteins and peptides in E. coli in an efficient manner. This appraisal provides an impression of expression technologies developed in recent times for enhanced production of heterologous proteins in E. coli which are of foremost importance for diverse applications in microbiology and biopharmaceutical production.

  4. Expression, Delivery and Function of Insecticidal Proteins Expressed by Recombinant Baculoviruses

    Science.gov (United States)

    Kroemer, Jeremy A.; Bonning, Bryony C.; Harrison, Robert L.

    2015-01-01

    Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification. PMID:25609310

  5. Preparative Purification of Recombinant Proteins: Current Status and Future Trends

    Directory of Open Access Journals (Sweden)

    Mayank Saraswat

    2013-01-01

    Full Text Available Advances in fermentation technologies have resulted in the production of increased yields of proteins of economic, biopharmaceutical, and medicinal importance. Consequently, there is an absolute requirement for the development of rapid, cost-effective methodologies which facilitate the purification of such products in the absence of contaminants, such as superfluous proteins and endotoxins. Here, we provide a comprehensive overview of a selection of key purification methodologies currently being applied in both academic and industrial settings and discuss how innovative and effective protocols such as aqueous two-phase partitioning, membrane chromatography, and high-performance tangential flow filtration may be applied independently of or in conjunction with more traditional protocols for downstream processing applications.

  6. Recent advances in recombinant protein-based malaria vaccines

    DEFF Research Database (Denmark)

    Draper, Simon J; Angov, Evelina; Horii, Toshihiro

    2015-01-01

    Plasmodium parasites are the causative agent of human malaria, and the development of a highly effective vaccine against infection, disease and transmission remains a key priority. It is widely established that multiple stages of the parasite's complex lifecycle within the human host and mosquito...... vector are susceptible to vaccine-induced antibodies. The mainstay approach to antibody induction by subunit vaccination has been the delivery of protein antigen formulated in adjuvant. Extensive efforts have been made in this endeavor with respect to malaria vaccine development, especially with regard......, with the prospects for the development of a highly effective multi-component/multi-stage/multi-antigen formulation seeming ever more likely. This review will focus on recent progress in protein vaccine design, development and/or clinical testing for a number of leading malaria antigens from the sporozoite...

  7. Preparation of recombinant coat protein of Prunus necrotic ringspot virus.

    Science.gov (United States)

    Petrzik, K; Mráz, I; Kubelková, D

    2001-02-01

    The coat protein (CP) gene of Prunus necrotic ringspot virus (PNRSV) was cloned into pET 16b vector and expressed in Escherichia coli. CP-enriched fractions were prepared from whole cell lysate by differential centrifugation. The fraction sedimenting at 20,000 x g for 30 mins was used for preparation of a rabbit antiserum to CP. This antiserum had a titer of 1:2048 and reacted in a double-antibody sandwich ELISA (DAS-ELISA).

  8. Tunable recombinant protein expression with E. coli in a mixed-feed environment.

    Science.gov (United States)

    Sagmeister, Patrick; Schimek, Clemens; Meitz, Andrea; Herwig, Christoph; Spadiut, Oliver

    2014-04-01

    Controlling the recombinant protein production rate in Escherichia coli is of utmost importance to ensure product quality and quantity. Up to now, only the genetic construct, introduced into E. coli, and the specific growth rate of the culture were used to influence and stir the productivity. However, bioprocess technological means to control or even tune the productivity of E. coli are scarce. Here, we present a novel method for the process-technological control over the recombinant protein expression rate in E. coli. A mixed-feed fed-batch bioprocess based on the araBAD promoter expression system using both D-glucose and L-arabinose as assimilable C-sources was designed. Using the model product green fluorescent protein, we show that the specific product formation rate can be efficiently tuned even on the cellular level only via the uptake rate of L-arabinose. This novel approach introduces an additional degree of freedom for the design of recombinant bioprocesses with E. coli. We anticipate that the presented method will result in significant quality and robustness improvement as well as cost and process time reduction for recombinant bacterial bioprocesses in the future.

  9. Construction of green fluorescent protein-tagged recombinant iridovirus to assess viral replication.

    Science.gov (United States)

    Huang, Youhua; Huang, Xiaohong; Cai, Jia; Ye, Fuzhou; Guan, Liya; Liu, Hong; Qin, Qiwei

    2011-09-01

    Green fluorescent protein-tagged recombinant virus has been successfully applied to observing the infective dynamics and evaluating viral replication. Here, we identified soft-shelled turtle iridovirus (STIV) ORF55 as an envelope protein (VP55), and developed a recombinant STIV expressing an enhanced green fluorescent protein (EGFP) fused to VP55 (EGFP-STIV). Recombinant EGFP-STIV shared similar single-step growth curves and ultrastructural morphology with wild type STIV (wt-STIV). The green fluorescence distribution during EGFP-STIV infection was consistent with the intracellular distribution of VP55 which was mostly co-localized with virus assembly sites. Furthermore, EGFP-STIV could be used to evaluate viral replication conveniently under drug treatment, and the result showed that STIV replication was significantly inhibited after the addition of antioxidant pyrrolidine dithiocarbamate (PDTC). Thus, the EGFP-tagged recombinant iridovirus will not only be useful for further investigations on the viral replicative dynamics, but also provide an alternative simple strategy to screen for antiviral substances. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. Multiplexed expression and screening for recombinant protein production in mammalian cells

    Directory of Open Access Journals (Sweden)

    McCafferty John

    2006-12-01

    Full Text Available Abstract Background A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner. Results A nested PCR approach was used to clone 21 extracellular domains of mouse receptors as CD4 fusions within a mammalian GATEWAY expression vector system. Following transient transfection, HEK-293E cells grown in 2 ml cultures in 24-deep well blocks showed similar growth kinetics, viability and recombinant protein expression profiles, to those grown in 50 ml shake flask cultures as judged by western blotting. Following optimisation, fluorescent dot blot analysis of transfection supernatants was shown to be a rapid method for analysing protein expression yielding similar results as western blot analysis. Addition of urea enhanced the binding of glycoproteins to a nitrocellulose membrane. A good correlation was observed between the results of a plate based small scale transient transfection dot blot pre-screen and successful purification of proteins expressed at the 50 ml scale. Conclusion The combination of small scale multi-well plate culture and dot blotting described here will allow the multiplex analysis of different mammalian expression experiments enabling a faster identification of high yield expression constructs or conditions prior to large scale protein production. The methods for parallel GATEWAY cloning and expression of multiple constructs in cell

  11. Efficient sortase-mediated N-terminal labeling of TEV protease cleaved recombinant proteins.

    Science.gov (United States)

    Sarpong, Kwabena; Bose, Ron

    2017-03-15

    A major challenge in attaching fluorophores or other handles to proteins is the availability of a site-specific labeling strategy that provides stoichiometric modification without compromising protein integrity. We developed a simple approach that combines TEV protease cleavage, sortase modification and affinity purification to N-terminally label proteins. To achieve stoichiometrically-labeled protein, we included a short affinity tag in the fluorophore-containing peptide for post-labeling purification of the modified protein. This strategy can be easily applied to any recombinant protein with a TEV site and we demonstrate this on Epidermal Growth Factor Receptor (EGFR) and Membrane Scaffold Protein (MSP) constructs. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Nuclear Engineering of Microalgae for High Yield Secretion of Recombinant Proteins

    DEFF Research Database (Denmark)

    Ramos Martinez, Erick Miguel

    biotechnology hosts including safety, metabolic diversity, scalability, sustainability and low production cost. Over the past decades, considerable improvement has been made to express and secrete recombinant proteins in high levels: however current yields are still low. The first research project presented...... to the glycomodules, accumulation of a fusion protein was dramatically increased by up to 12 folds, with the maximum yield of 15 mg L-1. Characterization of the secreted Venus showed the presence of glycosylations and increased resistance to proteolytic degradation. The results from this thesis demonstrate...... the potential of microalgae as a cell factory for secretion of recombinant proteins. The second research project presented in this thesis aimed to establish a new robust method to allow in vivo measurements of metabolic enzyme activities in cyanobacteria, with a hope that the method would facilitate further...

  13. Cross-system excision of chaperone-mediated proteolysis in chaperone-assisted recombinant protein production

    Science.gov (United States)

    Martínez-Alonso, Mónica; Villaverde, Antonio

    2010-01-01

    Main Escherichia coli cytosolic chaperones such as DnaK are key components of the control quality network designed to minimize the prevalence of polypeptides with aberrant conformations. This is achieved by both favoring refolding activities but also stimulating proteolytic degradation of folding reluctant species. This last activity is responsible for the decrease of the proteolytic stability of recombinant proteins when co-produced along with DnaK, where an increase in solubility might be associated to a decrease in protein yield. However, when DnaK and its co-chaperone DnaJ are co-produced in cultured insect cells or whole insect larvae (and expectedly, in other heterologous hosts), only positive, folding-related effects of these chaperones are observed, in absence of proteolysis-mediated reduction of recombinant protein yield. PMID:21326941

  14. Crystallization and preliminary crystallographic analysis of recombinant immunoglobulin G-binding protein from Streptococcus suis

    Energy Technology Data Exchange (ETDEWEB)

    Khan, Abdul Hamid; Chu, Fuliang; Feng, Youjun; Zhang, Qinagmin [Center for Molecular Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 (China); Qi, Jianxun [Institute of Physics, Chinese Academy of Sciences, Beijing 100080 (China); Gao, George Fu, E-mail: gaof@im.ac.cn [Center for Molecular Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 (China)

    2008-08-01

    Crystallization of recombinant IgG-binding protein expressed in Escherichia coli using the hanging-drop vapour-diffusion method is described. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 38.98, b = 43.94, c = 78.17 Å. Streptococcus suis, an important zoonotic pathogen, expresses immunoglobulin G-binding protein, which is thought to be helpful to the organism in eluding the host defence system. Recombinant IgG-binding protein expressed in Escherichia coli has been crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 38.98, b = 43.94, c = 78.17 Å and one molecule in the asymmetric unit. Diffraction data were collected to 2.60 Å resolution.

  15. Secretion of a recombinant protein without a signal peptide by the exocrine glands of transgenic rabbits.

    Directory of Open Access Journals (Sweden)

    Andrea Kerekes

    Full Text Available Transgenic rabbits carrying mammary gland specific gene constructs are extensively used for excreting recombinant proteins into the milk. Here, we report refined phenotyping of previously generated Venus transposon-carrying transgenic rabbits with particular emphasis on the secretion of the reporter protein by exocrine glands, such as mammary, salivary, tear and seminal glands. The Sleeping Beauty (SB transposon transgenic construct contains the Venus fluorophore cDNA, but without a signal peptide for the secretory pathway, driven by the ubiquitous CAGGS (CAG promoter. Despite the absence of a signal peptide, the fluorophore protein was readily detected in milk, tear, saliva and seminal fluids. The expression pattern was verified by Western blot analysis. Mammary gland epithelial cells of SB-CAG-Venus transgenic lactating does also showed Venus-specific expression by tissue histology and fluorescence microscopy. In summary, the SB-CAG-Venus transgenic rabbits secrete the recombinant protein by different glands. This finding has relevance not only for the understanding of the biological function of exocrine glands, but also for the design of constructs for expression of recombinant proteins in dairy animals.

  16. Tobacco BY-2 Media Component Optimization for a Cost-Efficient Recombinant Protein Production.

    Science.gov (United States)

    Häkkinen, Suvi T; Reuter, Lauri; Nuorti, Ninni; Joensuu, Jussi J; Rischer, Heiko; Ritala, Anneli

    2018-01-01

    Plant cells constitute an attractive platform for production of recombinant proteins as more and more animal-free products and processes are desired. One of the challenges in using plant cells as production hosts has been the costs deriving from expensive culture medium components. In this work, the aim was to optimize the levels of most expensive components in the nutrient medium without compromising the accumulation of biomass and recombinant protein yields. Wild-type BY-2 culture and transgenic tobacco BY-2 expressing green fluorescent protein-Hydrophobin I (GFP-HFBI) fusion protein were used to determine the most inexpensive medium composition. One particularly high-accumulating BY-2 clone, named 'Hulk,' produced 1.1 ± 0.2 g/l GFP-HFBI in suspension and kept its high performance during prolonged subculturing. In addition, both cultures were successfully cryopreserved enabling truly industrial application of this plant cell host. With the optimized culture medium, 43-55% cost reduction with regard to biomass and up to 69% reduction with regard to recombinant protein production was achieved.

  17. Methods for Using Small Non-Coding RNAs to Improve Recombinant Protein Expression in Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Sarah Inwood

    2018-01-01

    Full Text Available The ability to produce recombinant proteins by utilizing different “cell factories” revolutionized the biotherapeutic and pharmaceutical industry. Chinese hamster ovary (CHO cells are the dominant industrial producer, especially for antibodies. Human embryonic kidney cells (HEK, while not being as widely used as CHO cells, are used where CHO cells are unable to meet the needs for expression, such as growth factors. Therefore, improving recombinant protein expression from mammalian cells is a priority, and continuing effort is being devoted to this topic. Non-coding RNAs are RNA segments that are not translated into a protein and often have a regulatory role. Since their discovery, major progress has been made towards understanding their functions. Non-coding RNA has been investigated extensively in relation to disease, especially cancer, and recently they have also been used as a method for engineering cells to improve their protein expression capability. In this review, we provide information about methods used to identify non-coding RNAs with the potential of improving recombinant protein expression in mammalian cell lines.

  18. Improved Method for the Incorporation of Heme Cofactors into Recombinant Proteins Using Escherichia coli Nissle 1917.

    Science.gov (United States)

    Fiege, Kerstin; Querebillo, Christine Joy; Hildebrandt, Peter; Frankenberg-Dinkel, Nicole

    2018-05-15

    Recombinant production of heme proteins in Escherichia coli is often limited by the availability of heme in the host. Therefore, several methods, including the reconstitution of heme proteins after production but prior to purification or the HPEX system, conferring the ability to take up external heme have been developed and used in the past. Here we describe the use of the apathogenic E. coli strain Nissle 1917 (EcN) as a suitable host for the recombinant production of heme proteins. EcN has an advantage over commonly used lab strains in that it is able to take up heme from the environment through the heme receptor ChuA. Expression of several heme proteins from different prokaryotic sources led to high yield and quantitative incorporation of the cofactor when heme was supplied in the growth medium. Comparative UV-vis and resonance Raman measurements revealed that the method employed has significant influence on heme coordination with the EcN system representing the most native situation. Therefore, the use of EcN as a host for recombinant heme protein production represents an inexpensive and straightforward method to facilitate further investigations of structure and function.

  19. Secretion of a recombinant protein without a signal peptide by the exocrine glands of transgenic rabbits.

    Science.gov (United States)

    Kerekes, Andrea; Hoffmann, Orsolya Ivett; Iski, Gergely; Lipták, Nándor; Gócza, Elen; Kues, Wilfried A; Bősze, Zsuzsanna; Hiripi, László

    2017-01-01

    Transgenic rabbits carrying mammary gland specific gene constructs are extensively used for excreting recombinant proteins into the milk. Here, we report refined phenotyping of previously generated Venus transposon-carrying transgenic rabbits with particular emphasis on the secretion of the reporter protein by exocrine glands, such as mammary, salivary, tear and seminal glands. The Sleeping Beauty (SB) transposon transgenic construct contains the Venus fluorophore cDNA, but without a signal peptide for the secretory pathway, driven by the ubiquitous CAGGS (CAG) promoter. Despite the absence of a signal peptide, the fluorophore protein was readily detected in milk, tear, saliva and seminal fluids. The expression pattern was verified by Western blot analysis. Mammary gland epithelial cells of SB-CAG-Venus transgenic lactating does also showed Venus-specific expression by tissue histology and fluorescence microscopy. In summary, the SB-CAG-Venus transgenic rabbits secrete the recombinant protein by different glands. This finding has relevance not only for the understanding of the biological function of exocrine glands, but also for the design of constructs for expression of recombinant proteins in dairy animals.

  20. Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein.

    Science.gov (United States)

    Bertagnoli, S; Gelfi, J; Le Gall, G; Boilletot, E; Vautherot, J F; Rasschaert, D; Laurent, S; Petit, F; Boucraut-Baralon, C; Milon, A

    1996-08-01

    Two myxoma virus-rabbit hemorrhagic disease virus (RHDV) recombinant viruses were constructed with the SG33 strain of myxoma virus to protect rabbits against myxomatosis and rabbit viral hemorrhagic disease. These recombinant viruses expressed the RHDV capsid protein (VP60). The recombinant protein, which is 60 kDa in size, was antigenic, as revealed by its reaction in immunoprecipitation with antibodies raised against RHDV. Both recombinant viruses induced high levels of RHDV- and myxoma virus-specific antibodies in rabbits after immunization. Inoculations by the intradermal route protected animals against virulent RHDV and myxoma virus challenges.

  1. Purification and characterization of recombinant protein kinase CK2 from Zea mays expressed in Escherichia coli

    DEFF Research Database (Denmark)

    Riera, Marta; Pages, Montserrat; Issinger, Olaf Georg

    2003-01-01

    Recombinant protein kinase subunits rmCK2alpha-1 and rmCK2beta-1 from Zea mays were expressed separately in Escherichia coli and assembled to a fully active tetrameric holoenzyme complex in vitro. The obtained maize holoenzyme was purified to homogeneity, biochemically characterized, and compared...... to CK2 from human. Kinetic measurements of the recombinant maize holoenzyme (rmCK2) revealed k(cat) values for ATP and GTP of 4 and 2s(-1), respectively; whereas the recombinant maize catalytic subunit showed almost equal values for ATP and GTP, i.e., ca. 0.8s(-1). A comparison of the k(cat)/K(m) ratio...

  2. Arabidopsis PCH2 Mediates Meiotic Chromosome Remodeling and Maturation of Crossovers.

    Directory of Open Access Journals (Sweden)

    Christophe Lambing

    2015-07-01

    Full Text Available Meiotic chromosomes are organized into linear looped chromatin arrays by a protein axis localized along the loop-bases. Programmed remodelling of the axis occurs during prophase I of meiosis. Structured illumination microscopy (SIM has revealed dynamic changes in the chromosome axis in Arabidopsis thaliana and Brassica oleracea. We show that the axis associated protein ASY1 is depleted during zygotene concomitant with synaptonemal complex (SC formation. Study of an Atpch2 mutant demonstrates this requires the conserved AAA+ ATPase, PCH2, which localizes to the sites of axis remodelling. Loss of PCH2 leads to a failure to deplete ASY1 from the axes and compromizes SC polymerisation. Immunolocalization of recombination proteins in Atpch2 indicates that recombination initiation and CO designation during early prophase I occur normally. Evidence suggests that CO interference is initially functional in the mutant but there is a defect in CO maturation following designation. This leads to a reduction in COs and a failure to form COs between some homologous chromosome pairs leading to univalent chromosomes at metaphase I. Genetic analysis reveals that CO distribution is also affected in some chromosome regions. Together these data indicate that the axis remodelling defect in Atpch2 disrupts normal patterned formation of COs.

  3. High-level expression of soluble recombinant proteins in Escherichia coli using an HE-maltotriose-binding protein fusion tag.

    Science.gov (United States)

    Han, Yingqian; Guo, Wanying; Su, Bingqian; Guo, Yujie; Wang, Jiang; Chu, Beibei; Yang, Guoyu

    2018-02-01

    Recombinant proteins are commonly expressed in prokaryotic expression systems for large-scale production. The use of genetically engineered affinity and solubility enhancing fusion proteins has increased greatly in recent years, and there now exists a considerable repertoire of these that can be used to enhance the expression, stability, solubility, folding, and purification of their fusion partner. Here, a modified histidine tag (HE) used as an affinity tag was employed together with a truncated maltotriose-binding protein (MBP; consisting of residues 59-433) from Pyrococcus furiosus as a solubility enhancing tag accompanying a tobacco etch virus protease-recognition site for protein expression and purification in Escherichia coli. Various proteins tagged at the N-terminus with HE-MBP(Pyr) were expressed in E. coli BL21(DE3) cells to determine expression and solubility relative to those tagged with His6-MBP or His6-MBP(Pyr). Furthermore, four HE-MBP(Pyr)-fused proteins were purified by immobilized metal affinity chromatography to assess the affinity of HE with immobilized Ni 2+ . Our results showed that HE-MBP(Pyr) represents an attractive fusion protein allowing high levels of soluble expression and purification of recombinant protein in E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. A systematic investigation of production of synthetic prions from recombinant prion protein.

    Science.gov (United States)

    Schmidt, Christian; Fizet, Jeremie; Properzi, Francesca; Batchelor, Mark; Sandberg, Malin K; Edgeworth, Julie A; Afran, Louise; Ho, Sammy; Badhan, Anjna; Klier, Steffi; Linehan, Jacqueline M; Brandner, Sebastian; Hosszu, Laszlo L P; Tattum, M Howard; Jat, Parmjit; Clarke, Anthony R; Klöhn, Peter C; Wadsworth, Jonathan D F; Jackson, Graham S; Collinge, John

    2015-12-01

    According to the protein-only hypothesis, infectious mammalian prions, which exist as distinct strains with discrete biological properties, consist of multichain assemblies of misfolded cellular prion protein (PrP). A critical test would be to produce prion strains synthetically from defined components. Crucially, high-titre 'synthetic' prions could then be used to determine the structural basis of infectivity and strain diversity at the atomic level. While there have been multiple reports of production of prions from bacterially expressed recombinant PrP using various methods, systematic production of high-titre material in a form suitable for structural analysis remains a key goal. Here, we report a novel high-throughput strategy for exploring a matrix of conditions, additives and potential cofactors that might generate high-titre prions from recombinant mouse PrP, with screening for infectivity using a sensitive automated cell-based bioassay. Overall, approximately 20,000 unique conditions were examined. While some resulted in apparently infected cell cultures, this was transient and not reproducible. We also adapted published methods that reported production of synthetic prions from recombinant hamster PrP, but again did not find evidence of significant infectious titre when using recombinant mouse PrP as substrate. Collectively, our findings are consistent with the formation of prion infectivity from recombinant mouse PrP being a rare stochastic event and we conclude that systematic generation of prions from recombinant PrP may only become possible once the detailed structure of authentic ex vivo prions is solved. © 2015 The Authors.

  5. Simple and Efficient Purification of Recombinant Proteins Using the Heparin-Binding Affinity Tag.

    Science.gov (United States)

    Jayanthi, Srinivas; Gundampati, Ravi Kumar; Kumar, Thallapuranam Krishnaswamy Suresh

    2017-11-01

    Heparin, a member of the glycosaminoglycan family, is known to interact with more than 400 different types of proteins. For the past few decades, significant progress has been made to understand the molecular details involved in heparin-protein interactions. Based on the structural knowledge available from the FGF1-heparin interaction studies, we have designed a novel heparin-binding peptide (HBP) affinity tag that can be used for the simple, efficient, and cost-effective purification of recombinant proteins of interest. HBP-tagged fusion proteins can be purified by heparin Sepharose affinity chromatography using a simple sodium chloride gradient to elute the bound fusion protein. In addition, owing to the high density of positive charges on the HBP tag, recombinant target proteins are preferably expressed in their soluble forms. The purification of HBP-fusion proteins can also be achieved in the presence of chemical denaturants, including urea. Additionally, polyclonal antibodies raised against the affinity tag can be used to detect HBP-fused target proteins with high sensitivity. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  6. Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins.

    Science.gov (United States)

    Kleine Büning, Maximiliane; Meyer, Denise; Austermann-Busch, Sophia; Roman-Sosa, Gleyder; Rümenapf, Tillmann; Becher, Paul

    2017-04-01

    RNA recombination is a major driving force for the evolution of RNA viruses and is significantly implicated in the adaptation of viruses to new hosts, changes of virulence, as well as in the emergence of new viruses including drug-resistant and escape mutants. However, the molecular details of recombination in animal RNA viruses are only poorly understood. In order to determine whether viral RNA recombination depends on translation of viral proteins, a nonreplicative recombination system was established which is based on cotransfection of cells with synthetic bovine viral diarrhea virus (family Flaviviridae) RNA genome fragments either lacking the internal ribosome entry site required for cap-independent translation or lacking almost the complete polyprotein coding region. The emergence of a number of recombinant viruses demonstrated that IRES-mediated translation of viral proteins is dispensable for efficient recombination and suggests that RNA recombination can occur in the absence of viral proteins. Analyses of 58 independently emerged viruses led to the detection of recombinant genomes with duplications, deletions and insertions in the 5' terminal region of the open reading frame, leading to enlarged core fusion proteins detectable by Western blot analysis. This demonstrates a remarkable flexibility of the pestivirus core protein. Further experiments with capped and uncapped genome fragments containing a luciferase gene for monitoring the level of protein translation revealed that even a ∼1,000-fold enhancement of translation of viral proteins did not increase the frequency of RNA recombination. Taken together, this study highlights that nonreplicative RNA recombination does not require translation of viral proteins. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  7. Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins

    Science.gov (United States)

    Kleine Büning, Maximiliane; Meyer, Denise; Austermann-Busch, Sophia; Roman-Sosa, Gleyder; Rümenapf, Tillmann

    2017-01-01

    RNA recombination is a major driving force for the evolution of RNA viruses and is significantly implicated in the adaptation of viruses to new hosts, changes of virulence, as well as in the emergence of new viruses including drug-resistant and escape mutants. However, the molecular details of recombination in animal RNA viruses are only poorly understood. In order to determine whether viral RNA recombination depends on translation of viral proteins, a nonreplicative recombination system was established which is based on cotransfection of cells with synthetic bovine viral diarrhea virus (family Flaviviridae) RNA genome fragments either lacking the internal ribosome entry site required for cap-independent translation or lacking almost the complete polyprotein coding region. The emergence of a number of recombinant viruses demonstrated that IRES-mediated translation of viral proteins is dispensable for efficient recombination and suggests that RNA recombination can occur in the absence of viral proteins. Analyses of 58 independently emerged viruses led to the detection of recombinant genomes with duplications, deletions and insertions in the 5′ terminal region of the open reading frame, leading to enlarged core fusion proteins detectable by Western blot analysis. This demonstrates a remarkable flexibility of the pestivirus core protein. Further experiments with capped and uncapped genome fragments containing a luciferase gene for monitoring the level of protein translation revealed that even a ∼1,000-fold enhancement of translation of viral proteins did not increase the frequency of RNA recombination. Taken together, this study highlights that nonreplicative RNA recombination does not require translation of viral proteins. PMID:28338950

  8. Bovine Pancreatic Trypsin Inhibitor-Trypsin Complex as a Detection System for Recombinant Proteins

    Science.gov (United States)

    Borjigin, Jimo; Nathans, Jeremy

    1993-01-01

    Bovine pancreatic trypsin inhibitor (BPTI) binds to trypsin and anhydrotrypsin (an enzymatically inactive derivative of trypsin) with affinities of 6 x 10-14 and 1.1 x 10-13 M, respectively. We have taken advantage of the high affinity and specificity of this binding reaction to develop a protein tagging system in which biotinylated trypsin or biotinylated anhydrotrypsin is used as the reagent to detect recombinant fusion proteins into which BPTI has been inserted. Two proteins, opsin and growth hormone, were used as targets for insertional mutagenesis with BPTI. In each case, both domains of the fusion protein appear to be correctly folded. The fusion proteins can be specifically and efficiently detected by biotinylated trypsin or biotinylated anhydrotrypsin, as demonstrated by staining of transfected cells, protein blotting, affinity purification, and a mobility shift assay in SDS/polyacrylamide gels.

  9. Production of Brugia malayi BmSXP Recombinant Protein Expressed in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Khoo, T. K.

    2010-01-01

    Full Text Available A rapid antibody detection test is very useful for detection of lymphatic filariasis, especially for certification and surveillance of post-mass drug administration. One such kit, panLF RapidTM (commercialized by Malaysian BioDiagnostic Research Sdn. Bhd. had been developed in our laboratory for the detection of all species of filarial infections. It is based on the detection of anti-filarial IgG4 antibodies that react with recombinant Brugia malayi antigens, BmR1 and BmSXP. In this study, the growth of recombinant bacteria that produce BmSXP was optimized under shake flask fermentation for high yield of the recombinant antigen. The optimizations involved selection of suitable growth medium, IPTG concentration and induction time. The medium that yielded the highest biomass as well as total protein was Terrific Broth (TB medium, which is an undefined medium. Initiation of induction of protein expression was found to be best at mid-log phase (OD600 = 1.5, with IPTG concentration of 1.0 mM, and harvest time at 9 h post-induction. This study showed that under the optimized conditions, the shake flask culture produced 4 g/L biomass (dry cell weight of recombinant Escherichia coli BmSXP/pPROEXHTa/TOP10F’, which yielded 2.42 mg/L of purified BmSXP recombinant antigen. The purified antigen was analyzed by SDS-PAGE and the antigenicity of protein was confirmed by Western blot.

  10. Comparison of Immunoprotection of Leptospira Recombinant Proteins with conventional vaccine in experimental animals.

    Science.gov (United States)

    Parthiban, M; Kumar, S Senthil; Balachandran, C; Kumanan, K; Aarthi, K S; Nireesha, G

    2015-12-01

    Leptospirosis is a bacterial disease caused by bacteria of the genus Leptospira affecting humans and animals. Untreated leptospirosis may result in severe kidney damage, meningitis, liver failure, respiratory distress, and even death. Virulent leptospirosis can rapidly enter kidney fibroblasts and induce a programmed cell death. Thus, it is a challenge for immunologists to develop an effective and safe leptospirosis vaccine. Here, we compared the commercial canine leptospira vaccine and recombinant proteins (OmpL1 and LipL41) with and without adjuvant in terms of immune response and challenge studies in hamsters and immune response studies alone in experimental dogs. The outer membrane proteins viz., lipL41 and OmpL1 of leptospira interrogans serovars icterohaemorrhagiae were amplified. The primers were designed in such a way that amplified products of OmpL1 and lipL41 were ligated and cloned simultaneously into a single vector. The cloned products were expressed in E. coli BL21 cells. The immunoprotection studies were conducted for both recombinant proteins and commercial vaccine. The challenge experiment studies revealed that combination of both rLip41 and rOmpL1 and commercial vaccine gave 83% and 87% protection, respectively. Histopathological investigation revealed mild sub lethal changes were noticed in liver and kidney in commercially vaccinated group alone. The immune responses against recombinant leptospiral proteins were also demonstrated in dogs.

  11. Towards a more precise serological diagnosis of human tegumentary leishmaniasis using Leishmania recombinant proteins.

    Directory of Open Access Journals (Sweden)

    Ana Paula Souza

    Full Text Available BACKGROUND: Exposure to Leishmania induces a humoral immune response that can be used as a marker of parasite exposure. METHODOLOGY/PRINCIPAL FINDINGS: Herein, ELISA was used to screen sera from patients with Tegumentary Leishmaniasis (TL against different L. infantum-chagasi-derived recombinant proteins (rHSP70, rH2A, rH2B, rH3, rH4 and rKMP11. Among the recombinant proteins, rHSP70 and rH2A showed the best reactivity against human sera obtained from endemic areas of TL. Receiver-Operator Characteristics (ROC curve analysis was used to identify the effectiveness of these proteins for serodiagnosis of TL. ROC curves confirmed the superior performance of rHSP70 and rH2A, in comparison to the other tested recombinant proteins. Additionally, we evaluated the specificity of the response to rHSP70 and rH2A by testing sera obtained from patients with Chagas' disease, Tuberculosis, Leprosy or Systemic Lupus Erythematosus. In this case, rHSP70 displayed an increased ability to discriminate diseases, in comparison to SLA. CONCLUSION: Our results raise possibility of using rHSP70 for the serodiagnosis of TL.

  12. Single vector system for efficient N-myristoylation of recombinant proteins in E. coli.

    Directory of Open Access Journals (Sweden)

    Julian M Glück

    Full Text Available BACKGROUND: N-myristoylation is a crucial covalent modification of numerous eukaryotic and viral proteins that is catalyzed by N-myristoyltransferase (NMT. Prokaryotes are lacking endogenous NMT activity. Recombinant production of N-myristoylated proteins in E. coli cells can be achieved by coexpression of heterologous NMT with the target protein. In the past, dual plasmid systems were used for this purpose. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a single vector system for efficient coexpression of substrate and enzyme suitable for production of co- or posttranslationally modified proteins. The approach was validated using the HIV-1 Nef protein as an example. A simple and efficient protocol for production of highly pure and completely N-myristoylated Nef is presented. The yield is about 20 mg myristoylated Nef per liter growth medium. CONCLUSIONS/SIGNIFICANCE: The single vector strategy allows diverse modifications of target proteins recombinantly coexpressed in E. coli with heterologous enzymes. The method is generally applicable and provides large amounts of quantitatively processed target protein that are sufficient for comprehensive biophysical and structural studies.

  13. High-throughput purification of recombinant proteins using self-cleaving intein tags.

    Science.gov (United States)

    Coolbaugh, M J; Shakalli Tang, M J; Wood, D W

    2017-01-01

    High throughput methods for recombinant protein production using E. coli typically involve the use of affinity tags for simple purification of the protein of interest. One drawback of these techniques is the occasional need for tag removal before study, which can be hard to predict. In this work, we demonstrate two high throughput purification methods for untagged protein targets based on simple and cost-effective self-cleaving intein tags. Two model proteins, E. coli beta-galactosidase (βGal) and superfolder green fluorescent protein (sfGFP), were purified using self-cleaving versions of the conventional chitin-binding domain (CBD) affinity tag and the nonchromatographic elastin-like-polypeptide (ELP) precipitation tag in a 96-well filter plate format. Initial tests with shake flask cultures confirmed that the intein purification scheme could be scaled down, with >90% pure product generated in a single step using both methods. The scheme was then validated in a high throughput expression platform using 24-well plate cultures followed by purification in 96-well plates. For both tags and with both target proteins, the purified product was consistently obtained in a single-step, with low well-to-well and plate-to-plate variability. This simple method thus allows the reproducible production of highly pure untagged recombinant proteins in a convenient microtiter plate format. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Analysis of direct immobilized recombinant protein G on a gold surface

    International Nuclear Information System (INIS)

    Kim, Hyunhee; Kang, Da-Yeon; Goh, Hyun-Jeong; Oh, Byung-Keun; Singh, Ravindra P.; Oh, Soo-Min; Choi, Jeong-Woo

    2008-01-01

    Abstact: For the immobilization of IgG, various techniques such as chemical linker, thiolated protein G methods, and fragmentation of antibodies have been reported [Y.M. Bae, B.K. Oh, W. Lee, W.H. Lee, J.W. Choi, Biosensors Bioelectron. 21 (2005) 103; W. Lee, B.K. Oh, W.H. Lee, J.W. Choi, Colloids Surf. B-Biointerfaces, 40 (2005) 143; A.A. Karyakin, G.V. Presnova, M.Y. Rubtsova, A.M. Egorov, Anal. Chem. 72 (2000) 3805]. Here, we modified the immunoglobulin Fc-binding B-domain of protein G to contain two cysteine residues at its C-terminus by a genetic engineering technique. The resulting recombinant protein, RPGcys, retained IgG-binding activity in the same manner as native protein G. RPGcys was immobilized on a gold surface by strong affinity between thiol of cysteine and gold. The orientations of both IgG layers immobilized on the base recombinant protein Gs were analyzed by fluorescence microscope, atomic force microscope (AFM), and surface plasmon resonance (SPR). Our data revealed that IgG-binding activity of RPGcys on gold surface significantly increased in comparison to wild type of protein G (RPGwild), which was physically adsorbed due to absence of cysteine residue. Immobilization of highly oriented antibodies based on cysteine-modified protein G could be useful for the fabrication of immunosensor systems

  15. Pichia pastoris: a recombinant microfactory for antibodies and human membrane proteins.

    Science.gov (United States)

    Gonçalves, A M; Pedro, A Q; Maia, C; Sousa, F; Queiroz, J A; Passarinha, L A

    2013-05-01

    During the last few decades, it has become evident that the compatibility of the yeast biochemical environment with the ability to process and translate the RNA transcript, along with its capacity to modify a translated protein, are relevant requirements for selecting this host cell for protein expression in several pharmaceutical and clinical applications. In particular, Pichia pastoris is used as an industrial host for recombinant protein and metabolite production, showing a powerful capacity to meet required biomolecular target production levels in high-throughput assays for functional genomics and drug screening. In addition, there is a great advantage to using P. pastoris for protein secretion, even at high molecular weights, since the recovery and purification steps are simplified owing to relatively low levels of endogenous proteins in the extracellular medium. Clearly, no single microexpression system can provide all of the desired properties for human protein production. Moreover, chemical and physical bioprocess parameters, including culture medium formulation, temperature, pH, agitation, aeration rates, induction, and feeding strategies, can highly influence product yield and quality. In order to benefit from the currently available wide range of biosynthesis strategies using P. pastoris, this mini review focuses on the developments and technological fermentation achievements, providing both a comparative and an overall integration analysis. The main aim is to highlight the relevance and versatility of the P. pastoris biosystem to the design of more cost-effective microfactories to meet the increasing demands for recombinant membrane proteins and clinical antibodies for several therapeutic applications.

  16. Specific Genetic Immunotherapy Induced by Recombinant Vaccine Alpha-Fetoprotein-Heat Shock Protein 70 Complex

    Science.gov (United States)

    Wang, Xiaoping; Lin, Huanping; Wang, Qiaoxia

    Purposes: To construct a recombinant vaccine alpha-fetoprotein (AFP)-heat shock protein (HSP70) complex, and study its ability to induce specific CTL response and its protective effect against AFP-producing tumor. Material/Methods: A recombinant vaccine was constructed by conjugating mouse alpha-fetoprotein to heat shock protein 70. By way of intracutaneous injection, mice were primed and boosted with recombinant vaccine mAFP/HSP70, whereas single mAFP or HSP70 injection as controls. The ELISPOT and ELISA were used to measure the frequency of cells producing the cytokine IFN-γ in splenocytes and the level of anti-AFP antibody of serum from immunized mice respectively. In vivo tumor challenge were carried out to assess the immune effect of the recombinant vaccine. Results: By recombinant mAFP/HSP70 vaccine immunization, the results of ELISPOT and ELISA showed that the number of splenic cells producing IFN-γ and the level of anti-AFP antibody of serum were significantly higher in mAFP/HSP70 group than those in mAFP and HSP70 groups (108.50±11.70 IFN-γ spots/106 cells vs 41.60±10.40 IFN-γ spots/106 cells, 7.32±3.14 IFN-γ spots/106 cells, P<0.01; 156.32±10.42 μg/mL vs 66.52±7.35 μg/mL, 5.73±2.89 μg/mL, P<0.01). The tumor volume in mAFP/HSP70 group was significantly smaller than that in mAFP and HSP70 groups (42.44±7.14 mm3 vs 392.23±12.46 mm3, 838.63±13.84 mm3, P<0.01). Conclusions: The study further confirmed the function of heat shock protein 70's immune adjuvant. Sequential immunization with recombinant mAFP/HSP70 vaccine could generate effective antitumor immunity on AFP-producing tumor. The recombined mAFP/HSP70 vaccine may be suitable for serving as an immunotherapy for hepatocellular carcinoma.

  17. Higher accumulation of F1-V fusion recombinant protein in plants after induction of protein body formation.

    Science.gov (United States)

    Alvarez, M Lucrecia; Topal, Emel; Martin, Federico; Cardineau, Guy A

    2010-01-01

    Improving foreign protein accumulation is crucial for enhancing the commercial success of plant-based production systems since product yields have a major influence on process economics. Cereal grain evolved to store large amounts of proteins in tightly organized aggregates. In maize, gamma-Zein is the major storage protein synthesized by the rough endoplasmic reticulum (ER) and stored in specialized organelles called protein bodies (PB). Zera (gamma-Zein ER-accumulating domain) is the N-terminal proline-rich domain of gamma-zein that is sufficient to induce the assembly of PB formation. Fusion of the Zera domain to proteins of interest results in assembly of dense PB-like, ER-derived organelles, containing high concentration of recombinant protein. Our main goal was to increase recombinant protein accumulation in plants in order to enhance the efficiency of orally-delivered plant-made vaccines. It is well known that oral vaccination requires substantially higher doses than parental formulations. As a part of a project to develop a plant-made plague vaccine, we expressed our model antigen, the Yersinia pestis F1-V antigen fusion protein, with and without a fused Zera domain. We demonstrated that Zera-F1-V protein accumulation was at least 3x higher than F1-V alone when expressed in three different host plant systems: Ncotiana benthamiana, Medicago sativa (alfalfa) and Nicotiana tabacum NT1 cells. We confirmed the feasibility of using Zera technology to induce protein body formation in non-seed tissues. Zera expression and accumulation did not affect plant development and growth. These results confirmed the potential exploitation of Zera technology to substantially increase the accumulation of value-added proteins in plants.

  18. Recombination-stable multimeric green fluorescent protein for characterization of weak promoter outputs in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Rugbjerg, Peter; Knuf, Christoph; Förster, Jochen

    2015-01-01

    a less leaky Cu2+-inducible promoter based on CUP1. The basal expression level of the new promoter was approx. 61% below the wild-type CUP1 promoter, thus expanding the absolute range of Cu2+-based gene control. The stability of 3vGFP towards direct-repeat recombination was assayed in S. cerevisiae......Green fluorescent proteins (GFPs) are widely used for visualization of proteins to track localization and expression dynamics. However, phenotypically important processes can operate at too low expression levels for routine detection, i.e. be overshadowed by autofluorescence noise. While GFP...... functions well in translational fusions, the use of tandem GFPs to amplify fluorescence signals is currently avoided in Saccharomyces cerevisiae and many other microorganisms due to the risk of loop-out by direct-repeat recombination. We increased GFP fluorescence by translationally fusing three different...

  19. Advanced control of dissolved oxygen concentration in fed batch cultures during recombinant protein production.

    Science.gov (United States)

    Kuprijanov, A; Gnoth, S; Simutis, R; Lübbert, A

    2009-02-01

    Design and experimental validation of advanced pO(2) controllers for fermentation processes operated in the fed-batch mode are described. In most situations, the presented controllers are able to keep the pO(2) in fermentations for recombinant protein productions exactly on the desired value. The controllers are based on the gain-scheduling approach to parameter-adaptive proportional-integral controllers. In order to cope with the most often appearing distortions, the basic gain-scheduling feedback controller was complemented with a feedforward control component. This feedforward/feedback controller significantly improved pO(2) control. By means of numerical simulations, the controller behavior was tested and its parameters were determined. Validation runs were performed with three Escherichia coli strains producing different recombinant proteins. It is finally shown that the new controller leads to significant improvements in the signal-to-noise ratio of other key process variables and, thus, to a higher process quality.

  20. Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance

    Directory of Open Access Journals (Sweden)

    Cashman Kathleen A

    2008-06-01

    Full Text Available Abstract Background There is a significant requirement for the development and acquisition of reagents that will facilitate effective diagnosis, treatment, and prevention of Lassa fever. In this regard, recombinant Lassa virus (LASV proteins may serve as valuable tools in diverse antiviral applications. Bacterial-based systems were engineered for expression and purification of recombinant LASV nucleoprotein (NP, glycoprotein 1 (GP1, and glycoprotein 2 (GP2. Results Full-length NP and the ectodomains of GP1 and GP2 were generated as maltose-binding protein (MBP fusions in the Rosetta strains of Escherichia coli (E. coli using pMAL-c2x vectors. Average fusion protein yields per liter of culture for MBP-NP, MBP-GP1, and MBP-GP2 were 10 mg, 9 mg, and 9 mg, respectively. Each protein was captured from cell lysates using amylose resin, cleaved with Factor Xa, and purified using size-exclusion chromatography (SEC. Fermentation cultures resulted in average yields per liter of 1.6 mg, 1.5 mg, and 0.7 mg of purified NP, GP1 and GP2, respectively. LASV-specific antibodies in human convalescent sera specifically detected each of the purified recombinant LASV proteins, highlighting their utility in diagnostic applications. In addition, mouse hyperimmune ascitic fluids (MHAF against a panel of Old and New World arenaviruses demonstrated selective cross reactivity with LASV proteins in Western blot and enzyme-linked immunosorbent assay (ELISA. Conclusion These results demonstrate the potential for developing broadly reactive immunological assays that employ all three arenaviral proteins individually and in combination.

  1. Production of functional human insulin-like growth factor binding proteins (IGFBPs) using recombinant expression in HEK293 cells

    DEFF Research Database (Denmark)

    Wanscher, Anne Sofie Molsted; Williamson, Michael; Ebersole, Tasja Wainani

    2015-01-01

    on human proteins with therapeutic relevance is needed to design and process the next generation of protein therapeutics. In order to conduct structural and functional investigations large quantities of recombinant proteins are needed. However, finding a suitable recombinant production system for proteins...... and the final protein yields were between 1 and 12mg protein per liter culture media. The recombinant IGFBPs contained PTMs and exhibited high-affinity interactions with their natural ligands IGF-1 and IGF-2.......Insulin-like growth factor binding proteins (IGFBPs) display many functions in humans including regulation of the insulin-like growth factor (IGF) signaling pathway. The various roles of human IGFBPs make them attractive protein candidates in drug discovery. Structural and functional knowledge...

  2. Hijacked then lost in translation: the plight of the recombinant host cell in membrane protein structural biology projects.

    Science.gov (United States)

    Bill, Roslyn M; von der Haar, Tobias

    2015-06-01

    Membrane protein structural biology is critically dependent upon the supply of high-quality protein. Over the last few years, the value of crystallising biochemically characterised, recombinant targets that incorporate stabilising mutations has been established. Nonetheless, obtaining sufficient yields of many recombinant membrane proteins is still a major challenge. Solutions are now emerging based on an improved understanding of recombinant host cells; as a 'cell factory' each cell is tasked with managing limited resources to simultaneously balance its own growth demands with those imposed by an expression plasmid. This review examines emerging insights into the role of translation and protein folding in defining high-yielding recombinant membrane protein production in a range of host cells. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  3. Production, purification and oxidative folding of the mouse recombinant prion protein

    Czech Academy of Sciences Publication Activity Database

    Pavlíček, A.; Bednárová, Lucie; Holada, K.

    2007-01-01

    Roč. 52, č. 4 (2007), s. 391-397 ISSN 0015-5632 R&D Projects: GA ČR GD310/05/H533 Grant - others:GA ČR(CZ) GA310/04/0419 Institutional research plan: CEZ:AV0Z40550506 Keywords : recombinant prion protein * production * purification * folding Subject RIV: CE - Biochemistry Impact factor: 0.989, year: 2007 http://www.biomed.cas.cz/mbu/folia/

  4. Studies to Prevent Degradation of Recombinant Fc-Fusion Protein Expressed in Mammalian Cell Line and Protein Characterization

    Directory of Open Access Journals (Sweden)

    Sanjukta Chakrabarti

    2016-06-01

    Full Text Available Clipping of recombinant proteins is a major issue in animal cell cultures. A recombinant Fc-fusion protein, VEGFR1(D1–D3-Fc expressed in CHOK1SV GS-KO cells was observed to be undergoing clippings in lab scale cultures. Partial cleaving of expressed protein initiated early on in cell culture and was observed to increase over time in culture and also on storage. In this study, a few parameters were explored in a bid to inhibit clipping in the fusion protein The effects of culture temperature, duration of culture, the addition of an anti-clumping agent, ferric citrate and use of protease inhibitor cocktail on inhibition of proteolysis of the Fc fusion were studied. Lowering of culture temperature from 37 to 30 °C alone appears to be the best solution for reducing protein degradation from the quality, cost and regulatory points of view. The obtained Fc protein was characterized and found to be in its stable folded state, exhibiting a high affinity for its ligand and also biological and functional activities.

  5. Colocalization of somatic and meiotic double strand breaks near the Myc oncogene on mouse chromosome 15.

    Science.gov (United States)

    Ng, Siemon H; Maas, Sarah A; Petkov, Petko M; Mills, Kevin D; Paigen, Kenneth

    2009-10-01

    Both somatic and meiotic recombinations involve the repair of DNA double strand breaks (DSBs) that occur at preferred locations in the genome. Improper repair of DSBs during either mitosis or meiosis can lead to mutations, chromosomal aberration such as translocations, cancer, and/or cell death. Currently, no model exists that explains the locations of either spontaneous somatic DSBs or programmed meiotic DSBs or relates them to each other. One common class of tumorigenic translocations arising from DSBs is chromosomal rearrangements near the Myc oncogene. Myc translocations have been associated with Burkitt lymphoma in humans, plasmacytoma in mice, and immunocytoma in rats. Comparing the locations of somatic and meiotic DSBs near the mouse Myc oncogene, we demonstrated that the placement of these DSBs is not random and that both events clustered in the same short discrete region of the genome. Our work shows that both somatic and meiotic DSBs tend to occur in proximity to each other within the Myc region, suggesting that they share common originating features. It is likely that some regions of the genome are more susceptible to both somatic and meiotic DSBs, and the locations of meiotic hotspots may be an indicator of genomic regions more susceptible to DNA damage. (c) 2009 Wiley-Liss, Inc.

  6. Robotic high-throughput purification of affinity-tagged recombinant proteins.

    Science.gov (United States)

    Wiesler, Simone C; Weinzierl, Robert O J

    2015-01-01

    Affinity purification of recombinant proteins has become the method of choice to obtain good quantities and qualities of proteins for a variety of downstream biochemical applications. While manual or FPLC-assisted purification techniques are generally time-consuming and labor-intensive, the advent of high-throughput technologies and liquid handling robotics has simplified and accelerated this process significantly. Additionally, without the human factor as a potential source of error, automated purification protocols allow for the generation of large numbers of proteins simultaneously and under directly comparable conditions. The delivered material is ideal for activity comparisons of different variants of the same protein. Here, we present our strategy for the simultaneous purification of up to 24 affinity-tagged proteins for activity measurements in biochemical assays. The protocol described is suitable for the scale typically required in individual research laboratories.

  7. Main Strategies of Plant Expression System Glycoengineering for Producing Humanized Recombinant Pharmaceutical Proteins.

    Science.gov (United States)

    Rozov, S M; Permyakova, N V; Deineko, E V

    2018-03-01

    Most the pharmaceutical proteins are derived not from their natural sources, rather their recombinant analogs are synthesized in various expression systems. Plant expression systems, unlike mammalian cell cultures, combine simplicity and low cost of procaryotic systems and the ability for posttranslational modifications inherent in eucaryotes. More than 50% of all human proteins and more than 40% of the currently used pharmaceutical proteins are glycosylated, that is, they are glycoproteins, and their biological activity, pharmacodynamics, and immunogenicity depend on the correct glycosylation pattern. This review examines in detail the similarities and differences between N- and O-glycosylation in plant and mammalian cells, as well as the effect of plant glycans on the activity, pharmacokinetics, immunity, and intensity of biosynthesis of pharmaceutical proteins. The main current strategies of glycoengineering of plant expression systems aimed at obtaining fully humanized proteins for pharmaceutical application are summarized.

  8. Nested Inversion Polymorphisms Predispose Chromosome 22q11.2 to Meiotic Rearrangements

    NARCIS (Netherlands)

    Demaerel, Wolfram; Hestand, Matthew S.; Vergaelen, Elfi; Swillen, Ann; López-Sánchez, Marcos; Pérez-Jurado, Luis A.; McDonald-Mcginn, Donna M.; Zackai, Elaine; Emanuel, Beverly S.; Morrow, Bernice E.; Breckpot, Jeroen; Devriendt, Koenraad; Vermeesch, Joris R.; Antshel, Kevin M.; Arango, Celso; Armando, Marco; Bassett, Anne S.; Bearden, Carrie E.; Boot, Erik; Bravo-Sanchez, Marta; Breetvelt, Elemi; Busa, Tiffany; Butcher, Nancy J.; Campbell, Linda E.; Carmel, Miri; Chow, Eva W C; Crowley, T. Blaine; Cubells, Joseph; Cutler, David; Demaerel, Wolfram; Digilio, Maria Cristina; Duijff, Sasja; Eliez, Stephan; Emanuel, Beverly S.; Epstein, Michael P.; Evers, Rens; Fernandez Garcia-Moya, Luis; Fiksinski, Ania; Fraguas, David; Fremont, Wanda; Fritsch, Rosemarie; Garcia-Minaur, Sixto; Golden, Aaron; Gothelf, Doron; Guo, Tingwei; Gur, Ruben C.; Gur, Raquel E.; Heine-Suner, Damian; Hestand, Matthew; Hooper, Stephen R.; Kates, Wendy R.; Kushan, Leila; Laorden-Nieto, Alejandra; Maeder, Johanna; Marino, Bruno; Marshall, Christian R.; McCabe, Kathryn; McDonald-Mcginn, Donna M.; Michaelovosky, Elena; Morrow, Bernice E.; Moss, Edward; Mulle, Jennifer; Murphy, Declan; Murphy, Kieran C.; Murphy, Clodagh M.; Niarchou, Maria; Ornstein, Claudia; Owen, Michael J; Philip, Nicole; Repetto, Gabriela M.; Schneider, Maude; Shashi, Vandana; Simon, Tony J.; Swillen, Ann; Tassone, Flora; Unolt, Marta; Van Amelsvoort, Therese; van den Bree, Marianne B M; Van Duin, Esther; Vergaelen, Elfi; Vermeesch, Joris R.; Vicari, Stefano; Vingerhoets, Claudia; Vorstman, Jacob; Warren, Steve; Weinberger, Ronnie; Weisman, Omri; Weizman, Abraham; Zackai, Elaine; Zhang, Zhengdong; Zwick, Michael

    2017-01-01

    Inversion polymorphisms between low-copy repeats (LCRs) might predispose chromosomes to meiotic non-allelic homologous recombination (NAHR) events and thus lead to genomic disorders. However, for the 22q11.2 deletion syndrome (22q11.2DS), the most common genomic disorder, no such inversions have

  9. Structure of Mycobacterium tuberculosis RuvA, a protein involved in recombination

    International Nuclear Information System (INIS)

    Prabu, J. Rajan; Thamotharan, S.; Khanduja, Jasbeer Singh; Alipio, Emily Zabala; Kim, Chang-Yub; Waldo, Geoffrey S.; Terwilliger, Thomas C.; Segelke, Brent; Lekin, Tim; Toppani, Dominique; Hung, Li-Wei; Yu, Minmin; Bursey, Evan; Muniyappa, K.; Chandra, Nagasuma R.; Vijayan, M.

    2006-01-01

    RuvA, a protein from M. tuberculosis H37Rv involved in recombination, has been cloned, expressed, purified and analysed by X-ray crystallography. The process of recombinational repair is crucial for maintaining genomic integrity and generating biological diversity. In association with RuvB and RuvC, RuvA plays a central role in processing and resolving Holliday junctions, which are a critical intermediate in homologous recombination. Here, the cloning, purification and structure determination of the RuvA protein from Mycobacterium tuberculosis (MtRuvA) are reported. Analysis of the structure and comparison with other known RuvA proteins reveal an octameric state with conserved subunit–subunit interaction surfaces, indicating the requirement of octamer formation for biological activity. A detailed analysis of plasticity in the RuvA molecules has led to insights into the invariant and variable regions, thus providing a framework for understanding regional flexibility in various aspects of RuvA function

  10. Evaluation of Six Recombinant Proteins for Serological Diagnosis of Lyme Borreliosis in China.

    Science.gov (United States)

    Liu, Wei; Liu, Hui Xin; Zhang, Lin; Hou, Xue Xia; Wan, Kang Lin; Hao, Qin

    2016-05-01

    In this study, we evaluated the diagnostic efficiency of six recombinant proteins for the serodiagnosis of Lyme borreliosis (LB) and screened out the appropriate antigens to support the production of a Chinese clinical ELISA (enzyme-linked immunosorbent assay) kit for LB. Six recombinant antigens, Fla B.g, OspC B.a, OspC B.g, P39 B.g, P83 B.g, and VlsE B.a, were used for ELISA to detect serum antibodies in LB, syphilis, and healthy controls. The ELISA results were used to generate receiver operating characteristic (ROC) curves, and the sensitivity and specificity of each protein was evaluated. All recombinant proteins were evaluated and screened by using logistic regression models. Two IgG (VlsE and OspC B.g) and two IgM (OspC B.g and OspC B.a) antigens were left by the logistic regression model screened. VlsE had the highest specificity for syphilis samples in the IgG test (87.7%, Precombinant antigens, OspC B.g, OspC B.a, and VlsE B.a, were useful for ELISAs of LB. Additionally, the interaction between OspC B.a and Fla B.g should be examined in future research. Copyright © 2016 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  11. Generation of Recombinant Schmallenberg Virus Nucleocapsid Protein in Yeast and Development of Virus-Specific Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Justas Lazutka

    2014-01-01

    Full Text Available Schmallenberg virus (SBV, discovered in continental Europe in late 2011, causes mild clinical signs in adult ruminants, including diarrhoea and reduced milk yield. However, fetal infection can lead to severe malformation in newborn offspring. To develop improved reagents for SBV serology, a high-level yeast expression system was employed to produce recombinant SBV nucleocapsid (N protein. Recombinant SBV N protein was investigated as an antigen in SBV-specific IgG enzyme immunoassay and used for generation of monoclonal antibodies (MAbs. Yeast-expressed SBV N protein was reactive with anti-SBV IgG-positive cow serum specimens collected from different farms of Lithuania. After immunization of mice with recombinant SBV N protein, four MAbs were generated. The MAbs raised against recombinant SBV N protein reacted with native viral nucleocapsids in SBV-infected BHK cells by immunofluorescence assay. The reactivity of recombinant N protein with SBV-positive cow serum specimens and the ability of the MAbs to recognize virus-infected cells confirm the antigenic similarity between yeast-expressed SBV N protein and native viral nucleocapsids. Our study demonstrates that yeast expression system is suitable for high-level production of recombinant SBV N protein and provides the first evidence on the presence of SBV-specific antibodies in cow serum specimens collected in Lithuania.

  12. The Saccharomyces cerevisiae MUM2 gene interacts with the DNA replication machinery and is required for meiotic levels of double strand breaks.

    Science.gov (United States)

    Davis, L; Barbera, M; McDonnell, A; McIntyre, K; Sternglanz, R; Jin , Q; Loidl, J; Engebrecht, J

    2001-01-01

    The Saccharomyces cerevisiae MUM2 gene is essential for meiotic, but not mitotic, DNA replication and thus sporulation. Genetic interactions between MUM2 and a component of the origin recognition complex and polymerase alpha-primase suggest that MUM2 influences the function of the DNA replication machinery. Early meiotic gene expression is induced to a much greater extent in mum2 cells than in meiotic cells treated with the DNA synthesis inhibitor hydroxyurea. This result indicates that the mum2 meiotic arrest is downstream of the arrest induced by hydroxyurea and suggests that DNA synthesis is initiated in the mutant. Genetic analyses indicate that the recombination that occurs in mum2 mutants is dependent on the normal recombination machinery and on synaptonemal complex components and therefore is not a consequence of lesions created by incompletely replicated DNA. Both meiotic ectopic and allelic recombination are similarly reduced in the mum2 mutant, and the levels are consistent with the levels of meiosis-specific DSBs that are generated. Cytological analyses of mum2 mutants show that chromosome pairing and synapsis occur, although at reduced levels compared to wild type. Given the near-wild-type levels of meiotic gene expression, pairing, and synapsis, we suggest that the reduction in DNA replication is directly responsible for the reduced level of DSBs and meiotic recombination. PMID:11238403

  13. Tetratricopeptide-motif-mediated interaction of FANCG with recombination proteins XRCC3 and BRCA2.

    Science.gov (United States)

    Hussain, Shobbir; Wilson, James B; Blom, Eric; Thompson, Larry H; Sung, Patrick; Gordon, Susan M; Kupfer, Gary M; Joenje, Hans; Mathew, Christopher G; Jones, Nigel J

    2006-05-10

    Fanconi anaemia is an inherited chromosomal instability disorder characterised by cellular sensitivity to DNA interstrand crosslinkers, bone-marrow failure and a high risk of cancer. Eleven FA genes have been identified, one of which, FANCD1, is the breast cancer susceptibility gene BRCA2. At least eight FA proteins form a nuclear core complex required for monoubiquitination of FANCD2. The BRCA2/FANCD1 protein is connected to the FA pathway by interactions with the FANCG and FANCD2 proteins, both of which co-localise with the RAD51 recombinase, which is regulated by BRCA2. These connections raise the question of whether any of the FANC proteins of the core complex might also participate in other complexes involved in homologous recombination repair. We therefore tested known FA proteins for direct interaction with RAD51 and its paralogs XRCC2 and XRCC3. FANCG was found to interact with XRCC3, and this interaction was disrupted by the FA-G patient derived mutation L71P. FANCG was co-immunoprecipitated with both XRCC3 and BRCA2 from extracts of human and hamster cells. The FANCG-XRCC3 and FANCG-BRCA2 interactions did not require the presence of other FA proteins from the core complex, suggesting that FANCG also participates in a DNA repair complex that is downstream and independent of FANCD2 monoubiquitination. Additionally, XRCC3 and BRCA2 proteins co-precipitate in both human and hamster cells and this interaction requires FANCG. The FANCG protein contains multiple tetratricopeptide repeat motifs (TPRs), which function as scaffolds to mediate protein-protein interactions. Mutation of one or more of these motifs disrupted all of the known interactions of FANCG. We propose that FANCG, in addition to stabilising the FA core complex, may have a role in building multiprotein complexes that facilitate homologous recombination repair.

  14. Purification of Microbially Expressed Recombinant Proteins via a Dual ELP Split Intein System.

    Science.gov (United States)

    Shi, Changhua; Han, Tzu-Chiang; Wood, David W

    2017-01-01

    Fusions of elastin-like peptide (ELP) purification tags and self-cleaving inteins provide a powerful platform for purifying tagless recombinant proteins without the need for conventional packed-bed columns. A drawback to this method has been premature cleaving of the ELP tag during expression, before the purification procedure can take place. Here we demonstrate a split-intein method, where the self-cleaving intein is divided into two inactive segments during expression and purification. Spontaneous assembly of the purified intein segments then restores self-cleaving activity to deliver the tagless target protein.

  15. Outlook in the application of Chlamydomonas reinhardtii chloroplast as a platform for recombinant protein production.

    Science.gov (United States)

    Shamriz, Shabnam; Ofoghi, Hamideh

    Microalgae, also called microphytes, are a vast group of microscopic photosynthetic organisms living in aquatic ecosystems. Microalgae have attracted the attention of biotechnology industry as a platform for extracting natural products with high commercial value. During last decades, microalgae have been also used as cost-effective and easily scalable platform for the production of recombinant proteins with medical and industrial applications. Most progress in this field has been made with Chlamydomonas reinhardtii as a model organism mainly because of its simple life cycle, well-established genetics and ease of cultivation. However, due to the scarcity of existing infrastructure for commercial production and processing together with relatively low product yields, no recombinant products from C. reinhardtii have gained approval for commercial production and most of them are still in research and development. In this review, we focus on the chloroplast of C. reinhardtii as an algal recombinant expression platform and compare its advantages and disadvantages to other currently used expression systems. We then discuss the strategies for engineering the chloroplast of C. reinhardtii to produce recombinant cells and present a comprehensive overview of works that have used this platform for the expression of high-value products.

  16. Exaggerated inflammatory response after use of recombinant bone morphogenetic protein in recurrent unicameral bone cysts.

    Science.gov (United States)

    MacDonald, Kevin M; Swanstrom, Morgan M; McCarthy, James J; Nemeth, Blaise A; Guliani, Teresa A; Noonan, Kenneth J

    2010-03-01

    Recurrent unicameral bone cysts (UBCs) can result in significant morbidity during a child's physical and emotional development. Multiple treatment options are available and a review of the literature fails to clearly define the optimal treatment for UBCs. Recombinant bone morphogenetic protein (BMP) has been used with success in other disorders of poor bone formation. This manuscript is the first to report on the use of recombinant BMP in the treatment of UBCs. Three patients with recurrent UBCs underwent revision surgery with recombinant BMP. Radiographic and medical review was performed and is reported here. In these patients, the use of BMP failed to fully resolve their UBC; 2 patients had complete recurrence that required further surgery. In addition to poor radiographic results, all patients developed exaggerated inflammatory responses in the acute postoperative period. Each child developed clinically significant limb swelling and pain that mimicked infection. On the basis of our poor radiographic results and a paradoxical clinical result, we no longer recommend the use of recombinant BMP in the manner reported here for the treatment of recurrent UBCs. Level IV, case series.

  17. Construction and analysis of the transgenic carrot and celery plants expressing the recombinant thaumatin II protein

    Directory of Open Access Journals (Sweden)

    Luchakivska Yu. S.

    2015-08-01

    Full Text Available Aim To obtain the transgenic carrot and celery plants able to express recombinant thaumatin II in order to increase plant stress tolerance. Methods. Agrobacterium-mediated transformation of the carrot and celery seedlings was used for obtaining the transgenic plants. Presence and transcription of the transgene in plant tissues were proved by PCR and RT-PCR analysis. The plants were tested for the biotic stress tolerance by in vitro antifungal and antibacterial activity assays and for the salinity and osmotic stress tolerance by plant survival test in presence of NaCl and PEG in different concentrations. Results. Transgenic plants able to express recombinant thaumatin II gene (transcription proved for 60–100 % were obtained by agrobacterial transformation. The transgenic carrot plant extracts inhibited the growth of the studied phytopathogenic bacteria strains but exhibited no antifungal activity. Survival level of transgenic plants under the salinity and osmotic stress effect was definitely higher comparing to the untransgenic ones. The analysis of the photosynthetic pigment content in the transgenic carrot plants showed no significant difference of this parameter under salinity stress that may indicate a possible protective activity of the recombinant protein. Conclusions. The obtained in our study transgenic carrot and celery plants able to express the recombinant thaumatin II gene were characterized by antibacterial activity and increased tolerance to salinity and osmotic stress factors.

  18. Targeted disruption of exons 1 to 6 of the Fanconi Anemia group A gene leads to growth retardation, strain-specific microphthalmia, meiotic defects and primordial germ cell hypoplasia.

    Science.gov (United States)

    Wong, Jasmine C Y; Alon, Noa; Mckerlie, Colin; Huang, Jun R; Meyn, M Stephen; Buchwald, Manuel

    2003-08-15

    Fanconi Anemia (FA) is an autosomal recessive disorder characterized by cellular hypersensitivity to DNA cross-linking agents. Recent studies suggest that FA proteins share a common pathway with BRCA proteins. To study the in vivo role of the FA group A gene (Fanca), gene-targeting techniques were used to generate Fanca(tm1Hsc) mice in which Fanca exons 1-6 were replaced by a beta-galactosidase reporter construct. Fanca(tm1.1Hsc) mice were generated by Cre-mediated removal of the neomycin cassette in Fanca(tm1Hsc) mice. Fanca(tm1.1Hsc) homozygotes display FA-like phenotypes including growth retardation, microphthalmia and craniofacial malformations that are not found in other Fanca mouse models, and the genetic background affects manifestation of certain phenotypes. Both male and female mice homozygous for Fanca mutation exhibit hypogonadism, and homozygous females demonstrate premature reproductive senescence and an increased incidence of ovarian cysts. We showed that fertility defects in Fanca(tm1.1Hsc) homozygotes might be related to a diminished population of primordial germ cells (PGCs) during migration into the gonadal ridges. We also found a high level of Fanca expression in pachytene spermatocytes. Fanca(tm1Hsc) homozygous males exhibited an elevated frequency of mispaired meiotic chromosomes and increased apoptosis in germ cells, implicating a role for Fanca in meiotic recombination. However, the localization of Rad51, Brca1, Fancd2 and Mlh1 appeared normal on Fanca(tm1Hsc) homozygous meiotic chromosomes. Taken together, our results suggest that the FA pathway plays a role in the maintenance of reproductive germ cells and in meiotic recombination.

  19. Vaccination with recombinant heat shock protein 60 from Histoplasma capsulatum protects mice against pulmonary histoplasmosis.

    Science.gov (United States)

    Gomez, F J; Allendoerfer, R; Deepe, G S

    1995-07-01

    HIS-62 is a glycoprotein that has been isolated from the cell wall and cell membrane fraction of the pathogenic fungus Histoplasma capsulatum. It is a target of the cellular immune response to this fungus, and it protects mice against a lethal intravenous inoculum of H. capsulatum yeast cells. In this study, we cloned the gene encoding this antigen to reveal its biological nature and studied the immunological activity of recombinant antigen. The amino acid sequences of the NH2 terminus and internal peptides were obtained by Edman degradation. Degenerate oligonucleotides were used to isolate a gene fragment of HIS-62 by PCR. One 680-bp segment that corresponded to the known peptide sequence was amplified from H. capsulatum DNA. This DNA was used to screen a genomic library, and the full-length gene was isolated and sequenced. The deduced amino acid sequence of the gene demonstrated approximately 70 and approximately 50% identity to heat shock protein 60 (hsp 60) from Saccharomyces cerevisiae and hsp 60 from Escherichia coli, respectively. A cDNA was synthesized by reverse transcription PCR and was expressed in E. coli. Recombinant protein reacted with a monospecific polyclonal rabbit antiserum raised against native HIS-62, with monoclonal HIS-62-reactive T cells, and with splenocytes from mice immunized with viable yeast cells. Moreover, vaccination with the recombinant protein conferred protection in mice against a lethal intranasal inoculation with yeast cells. Thus, HIS-62 is a member of the hsp 60 family, and the recombinant hsp 60 is protective against pulmonary histoplasmosis in mice.

  20. Overexpression of the transcription factor Yap1 modifies intracellular redox conditions and enhances recombinant protein secretion

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    Marizela Delic

    2014-10-01

    Full Text Available Oxidative folding of secretory proteins in the endoplasmic reticulum (ER is a redox active process, which also impacts the redox conditions in the cytosol. As the transcription factor Yap1 is involved in the transcriptional response to oxidative stress, we investigate its role upon the production of secretory proteins, using the yeast Pichia pastoris as model, and report a novel important role of Yap1 during oxidative protein folding. Yap1 is needed for the detoxification of reactive oxygen species (ROS caused by increased oxidative protein folding. Constitutive co-overexpression of PpYAP1 leads to increased levels of secreted recombinant protein, while a lowered Yap1 function leads to accumulation of ROS and strong flocculation. Transcriptional analysis revealed that more than 150 genes were affected by overexpression of YAP1, in particular genes coding for antioxidant enzymes or involved in oxidation-reduction processes. By monitoring intracellular redox conditions within the cytosol and the ER using redox-sensitive roGFP1 variants, we could show that overexpression of YAP1 restores cellular redox conditions of protein-secreting P. pastoris by reoxidizing the cytosolic redox state to the levels of the wild type. These alterations are also reflected by increased levels of oxidized intracellular glutathione (GSSG in the YAP1 co-overexpressing strain. Taken together, these data indicate a strong impact of intracellular redox balance on the secretion of (recombinant proteins without affecting protein folding per se. Re-establishing suitable redox conditions by tuning the antioxidant capacity of the cell reduces metabolic load and cell stress caused by high oxidative protein folding load, thereby increasing the secretion capacity.

  1. Scale-up of hydrophobin-assisted recombinant protein production in tobacco BY-2 suspension cells.

    Science.gov (United States)

    Reuter, Lauri J; Bailey, Michael J; Joensuu, Jussi J; Ritala, Anneli

    2014-05-01

    Plant suspension cell cultures are emerging as an alternative to mammalian cells for production of complex recombinant proteins. Plant cell cultures provide low production cost, intrinsic safety and adherence to current regulations, but low yields and costly purification technology hinder their commercialization. Fungal hydrophobins have been utilized as fusion tags to improve yields and facilitate efficient low-cost purification by surfactant-based aqueous two-phase separation (ATPS) in plant, fungal and insect cells. In this work, we report the utilization of hydrophobin fusion technology in tobacco bright yellow 2 (BY-2) suspension cell platform and the establishment of pilot-scale propagation and downstream processing including first-step purification by ATPS. Green fluorescent protein-hydrophobin fusion (GFP-HFBI) induced the formation of protein bodies in tobacco suspension cells, thus encapsulating the fusion protein into discrete compartments. Cultivation of the BY-2 suspension cells was scaled up in standard stirred tank bioreactors up to 600 L production volume, with no apparent change in growth kinetics. Subsequently, ATPS was applied to selectively capture the GFP-HFBI product from crude cell lysate, resulting in threefold concentration, good purity and up to 60% recovery. The ATPS was scaled up to 20 L volume, without loss off efficiency. This study provides the first proof of concept for large-scale hydrophobin-assisted production of recombinant proteins in tobacco BY-2 cell suspensions. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  2. Use of recombinant purified protein derivative (PPD) antigens as specific skin test for tuberculosis.

    Science.gov (United States)

    Stavri, Henriette; Bucurenci, Nadia; Ulea, Irina; Costache, Adriana; Popa, Loredana; Popa, Mircea Ioan

    2012-11-01

    Purified protein derivative (PPD) is currently the only available skin test reagent used worldwide for the diagnosis of tuberculosis (TB). The aim of this study was to develop a Mycobacterium tuberculosis specific skin test reagent, without false positive results due to Bacillus Calmette-Guerin (BCG) vaccination using recombinant antigens. Proteins in PPD IC-65 were analyzed by tandem mass spectrometry and compared to proteins in M. tuberculosis culture filtrate; 54 proteins were found in common. Top candidates MPT64, ESAT 6, and CFP 10 were overexpressed in Escherichia coli expression strains and purified as recombinant proteins. To formulate optimal immunodiagnostic PPD cocktails, the antigens were evaluated by skin testing guinea pigs sensitized with M. tuberculosis H37Rv and BCG. For single antigens and a cocktail mixture of these antigens, best results were obtained using 3 μg/0.1 ml, equivalent to 105 TU (tuberculin units). Each animal was simultaneously tested with PPD IC-65, 2 TU/0.1 ml, as reference. Reactivity of the multi-antigen cocktail was greater than that of any single antigen. The skin test results were between 34.3 and 76.6 per cent the level of reactivity compared to that of the reference when single antigens were tested and 124 per cent the level of reactivity compared to the reference for the multi-antigen cocktail. Our results showed that this specific cocktail could represent a potential candidate for a new skin diagnostic test for TB.

  3. Engineer medium and feed for modulating N-glycosylation of recombinant protein production in CHO cell culture

    DEFF Research Database (Denmark)

    Fan, Yuzhou; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

    2017-01-01

    Chinese hamster ovary (CHO) cells have become the primary expression system for the production of complex recombinant proteins due to their long-term success in industrial scale production and generating appropriate protein N-glycans similar to that of humans. Control and optimization of protein N......-glycosylation is crucial, as the structure of N-glycans can largely influence both biological and physicochemical properties of recombinant proteins. Protein N-glycosylation in CHO cell culture can be controlled and tuned by engineering medium, feed, culture process, as well as genetic elements of the cell...

  4. Regulation of homologous recombination in eukaryotes

    OpenAIRE

    Heyer, Wolf-Dietrich; Ehmsen, Kirk T.; Liu, Jie

    2010-01-01

    Homologous recombination is required for accurate chromosome segregation during the first meiotic division and constitutes a key repair and tolerance pathway for complex DNA damage including DNA double-stranded breaks, interstrand crosslinks, and DNA gaps. In addition, recombination and replication are inextricably linked, as recombination recovers stalled and broken replication forks enabling the evolution of larger genomes/replicons. Defects in recombination lead to genomic instability and ...

  5. A Novel Recombinant DNA System for High Efficiency Affinity Purification of Proteins in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Brian H. Carrick

    2016-03-01

    Full Text Available Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs using homologous recombination of DNA fragments. Tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this, we have created a recombinant “CelTag” DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent could allow isolation of protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or nucleic acid-associated assemblies.

  6. Methods for efficient high-throughput screening of protein expression in recombinant Pichia pastoris strains.

    Science.gov (United States)

    Camattari, Andrea; Weinhandl, Katrin; Gudiminchi, Rama K

    2014-01-01

    The methylotrophic yeast Pichia pastoris is becoming one of the favorite industrial workhorses for protein expression. Due to the widespread use of integration vectors, which generates significant clonal variability, screening methods allowing assaying hundreds of individual clones are of particular importance. Here we describe methods to detect and analyze protein expression, developed in a 96-well format for high-throughput screening of recombinant P. pastoris strains. The chapter covers essentially three common scenarios: (1) an enzymatic assay for proteins expressed in the cell cytoplasm, requiring cell lysis; (2) a whole-cell assay for a fungal cytochrome P450; and (3) a nonenzymatic assay for detection and quantification of tagged protein secreted into the supernatant.

  7. [A study of recombinant human sestrin 1 and sestrin 2 proteins produced in a prokaryotic system].

    Science.gov (United States)

    Rai, N; Kumar, R; Haque, Md A; Hassan, Md I; Dey, S

    2017-01-01

    Sestrins are highly conserved stress-inducible proteins capable of suppressing the production of ROS and signalling through mTORC1. Here we report a study of human sestrin1 (sesn1) and sestrin2 (sesn2) proteins produced in a pET28^(+) vector based prokaryotic system. Mass spectrometry analysis, western blot and surface plasmon resonance (SPR) of affinity purified sesn1 and sesn2 proteins confirmed their identity; biophysical characteristics were observed using circular dichroism (CD) showing that sesn1 and sesn2 have a predominant α-helical structure. Here we describe a simple, one step purification process to purify a large amount of sestrin proteins with significant yield. Further study of recombinant human sestrins may further facilitate the understanding of their roles in eukaryotic cells.

  8. Recombinant expression, purification, and characterization of an acyl-CoA binding protein from Aspergillus oryzae.

    Science.gov (United States)

    Hao, Qing; Liu, Xiaoguang; Zhao, Guozhong; Jiang, Lu; Li, Ming; Zeng, Bin

    2016-03-01

    To characterize biochemically the lipid metabolism-regulating acyl-CoA binding protein (ACBP) from the industrially-important fungus Aspergillus oryzae. A full-length cDNA encoding a candidate ACBP from A. oryzae (AoACBP) was cloned and expressed in Escherichia coli as a maltose-binding protein (MBP) fusion protein. The MBP-AoACBP protein was purified by an amylose resin chromatography column. SDS-PAGE showed that MBP-AoACBP has an estimated molecular weight of 82 kDa. Microscale thermophoresis binding assay showed that the recombinant AoACBP displayed much greater affinity for palmitoyl-CoA (K d = 80 nM) than for myristoyl-CoA (K d = 510 nM), thus demonstrating the preference of AoACBP for long-chain acyl-CoA. The data support the identification of AoACBP as a long-chain ACBP in A. oryzae.

  9. Vaccinia virus recombinants expressing chimeric proteins of human immunodeficiency virus and gamma interferon are attenuated for nude mice.

    OpenAIRE

    Giavedoni, L D; Jones, L; Gardner, M B; Gibson, H L; Ng, C T; Barr, P J; Yilma, T

    1992-01-01

    We have developed a method for attenuating vaccinia virus recombinants by expressing a fusion protein of a lymphokine and an immunogen. Chimeric genes were constructed that coded for gamma interferon (IFN-gamma) and structural proteins of the human immunodeficiency virus type 1 (HIV-1). In this study, we describe the biological and immunological properties of vaccinia virus recombinants expressing chimeric genes of murine or human IFN-gamma with glycoprotein gp120, gag, and a fragment of gp41...

  10. Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins.

    Directory of Open Access Journals (Sweden)

    Nan Zhong

    Full Text Available We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP protocols.

  11. Reassessment of inclusion body-based production as a versatile opportunity for difficult-to-express recombinant proteins.

    Science.gov (United States)

    Hoffmann, Daniel; Ebrahimi, Mehrdad; Gerlach, Doreen; Salzig, Denise; Czermak, Peter

    2017-11-10

    The production of recombinant proteins in the microbial host Escherichia coli often results in the formation of cytoplasmic protein inclusion bodies (IBs). Proteins forming IBs are often branded as difficult-to-express, neglecting that IBs can be an opportunity for their production. IBs are resistant to proteolytic degradation and contain up to 90% pure recombinant protein, which does not interfere with the host metabolism. This is especially advantageous for host-toxic proteins like antimicrobial peptides (AMPs). IBs can be easily isolated by cell disruption followed by filtration and/or centrifugation, but conventional techniques for the recovery of soluble proteins from IBs are laborious. New approaches therefore simplify protein recovery by optimizing the production process conditions, and often include mild resolubilization methods that either increase the yield after refolding or avoid the necessity of refolding all together. For the AMP production, the IB-based approach is ideal, because these peptides often have simple structures and are easy to refold. The intentional IB production of almost every protein can be achieved by fusing recombinant proteins to pull-down tags. This review discusses the techniques available for IB-based protein production before considering technical approaches for the isolation of IBs from E. coli lysates followed by efficient protein resolubilization which ideally omits further refolding. The techniques are evaluated in terms of their suitability for the process-scale production and downstream processing of recombinant proteins and are discussed for AMP production as an example.

  12. A synthetic arabinose-inducible promoter confers high levels of recombinant protein expression in hyperthermophilic archaeon Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Peng, Nan; Deng, Ling; Mei, Yuxia

    2012-01-01

    Despite major progresses in genetic studies of hyperthermophilic archaea, recombinant protein production in these organisms always suffers from low yields and a robust expression system is still in great demand. Here we report a versatile vector that confers high levels of protein expression...... to remove the peptide tags from expressed recombinant proteins. While pEXA employed an araS promoter for protein expression, pSeSD utilized P(araS-SD), an araS derivative promoter carrying an engineered ribosome-binding site (RBS; a Shine-Dalgarno [SD] sequence). We found that P(araS-SD) directed high...... levels of target gene expression. More strikingly, N-terminal amino acid sequencing of recombinant proteins unraveled that the protein synthesized from pEXA-N-lacS lacked the designed 6×His tag and that translation initiation did not start at the ATG codon of the fusion gene. Instead, it started...

  13. Subdominant Outer Membrane Antigens in Anaplasma marginale: Conservation, Antigenicity, and Protective Capacity Using Recombinant Protein.

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    Deirdre R Ducken

    Full Text Available Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP extracts or a defined surface protein complex reproducibly induce protective immunity. However, there are several knowledge gaps limiting progress in vaccine development. First, are these OMPs conserved among the diversity of A. marginale strains circulating in endemic regions? Second, are the most highly conserved outer membrane proteins in the immunogens recognized by immunized and protected animals? Lastly, can this subset of OMPs recognized by antibody from protected vaccinates and conserved among strains recapitulate the protection of outer membrane vaccines? To address the first goal, genes encoding OMPs AM202, AM368, AM854, AM936, AM1041, and AM1096, major subdominant components of the outer membrane, were cloned and sequenced from geographically diverse strains and isolates. AM202, AM936, AM854, and AM1096 share 99.9 to 100% amino acid identity. AM1041 has 97.1 to 100% and AM368 has 98.3 to 99.9% amino acid identity. While all four of the most highly conserved OMPs were recognized by IgG from animals immunized with outer membranes, linked surface protein complexes, or unlinked surface protein complexes and shown to be protected from challenge, the highest titers and consistent recognition among vaccinates were to AM854 and AM936. Consequently, animals were immunized with recombinant AM854 and AM936 and challenged. Recombinant vaccinates and purified outer membrane vaccinates had similar IgG and IgG2 responses to both proteins. However, the recombinant vaccinates developed higher bacteremia after challenge as compared to adjuvant-only controls and outer membrane vaccinates. These results provide the first evidence that vaccination with specific antigens may exacerbate disease. Progressing from the protective capacity of outer membrane formulations to

  14. Recombinant Vaccinia Viruses Coding Transgenes of Apoptosis-Inducing Proteins Enhance Apoptosis But Not Immunogenicity of Infected Tumor Cells

    Science.gov (United States)

    Tkachenko, Anastasiya; Richter, Vladimir

    2017-01-01

    Genetic modifications of the oncolytic vaccinia virus (VV) improve selective tumor cell infection and death, as well as activation of antitumor immunity. We have engineered a double recombinant VV, coding human GM-CSF, and apoptosis-inducing protein apoptin (VV-GMCSF-Apo) for comparing with the earlier constructed double recombinant VV-GMCSF-Lact, coding another apoptosis-inducing protein, lactaptin, which activated different cell death pathways than apoptin. We showed that both these recombinant VVs more considerably activated a set of critical apoptosis markers in infected cells than the recombinant VV coding GM-CSF alone (VV-GMCSF-dGF): these were phosphatidylserine externalization, caspase-3 and caspase-7 activation, DNA fragmentation, and upregulation of proapoptotic protein BAX. However, only VV-GMCSF-Lact efficiently decreased the mitochondrial membrane potential of infected cancer cells. Investigating immunogenic cell death markers in cancer cells infected with recombinant VVs, we demonstrated that all tested recombinant VVs were efficient in calreticulin and HSP70 externalization, decrease of cellular HMGB1, and ATP secretion. The comparison of antitumor activity against advanced MDA-MB-231 tumor revealed that both recombinants VV-GMCSF-Lact and VV-GMCSF-Apo efficiently delay tumor growth. Our results demonstrate that the composition of GM-CSF and apoptosis-inducing proteins in the VV genome is very efficient tool for specific killing of cancer cells and for activation of antitumor immunity. PMID:28951871

  15. Localization of recombination proteins and Srs2 reveals anti-recombinase function in vivo

    DEFF Research Database (Denmark)

    Burgess, Rebecca C; Lisby, Michael; Altmannova, Veronika

    2009-01-01

    , and surprisingly, can form in the absence of Rad52 mediation. However, these Rad51 foci do not represent repair-proficient filaments, as determined by recombination assays. Antagonistic roles for Rad52 and Srs2 in Rad51 filament formation are also observed in vitro. Furthermore, we provide evidence that Srs2......Homologous recombination (HR), although an important DNA repair mechanism, is dangerous to the cell if improperly regulated. The Srs2 "anti-recombinase" restricts HR by disassembling the Rad51 nucleoprotein filament, an intermediate preceding the exchange of homologous DNA strands. Here, we...... removes Rad51 indiscriminately from DNA, while the Rad52 protein coordinates appropriate filament reformation. This constant breakdown and rebuilding of filaments may act as a stringent quality control mechanism during HR....

  16. Residual endotoxin contaminations in recombinant proteins are sufficient to activate human CD1c+ dendritic cells.

    Directory of Open Access Journals (Sweden)

    Harald Schwarz

    Full Text Available Many commercially available recombinant proteins are produced in Escherichia coli, and most suppliers guarantee contamination levels of less than 1 endotoxin unit (EU. When we analysed commercially available proteins for their endotoxin content, we found contamination levels in the same range as generally stated in the data sheets, but also some that were higher. To analyse whether these low levels of contamination have an effect on immune cells, we stimulated the monocytic cell line THP-1, primary human monocytes, in vitro differentiated human monocyte-derived dendritic cells, and primary human CD1c+ dendritic cells (DCs with very low concentrations of lipopolysaccharide (LPS; ranging from 0.002-2 ng/ml. We show that CD1c+ DCs especially can be activated by minimal amounts of LPS, equivalent to the levels of endotoxin contamination we detected in some commercially available proteins. Notably, the enhanced endotoxin sensitivity of CD1c+ DCs was closely correlated with high CD14 expression levels observed in CD1c+ DCs that had been maintained in cell culture medium for 24 hours. When working with cells that are particularly sensitive to LPS, even low endotoxin contamination may generate erroneous data. We therefore recommend that recombinant proteins be thoroughly screened for endotoxin contamination using the limulus amebocyte lysate test, fluorescence-based assays, or a luciferase based NF-κB reporter assay involving highly LPS-sensitive cells overexpressing TLR4, MD-2 and CD14.

  17. Recombinant Protein Production from TPO Gen Cloning and Expression for Early Detection of Autoimmune Thyroid Diseases

    Science.gov (United States)

    Aulanni'am, Aulanni'am; Kinasih Wuragil, Dyah; Wahono Soeatmadji, Djoko; Zulkarnain; Marhendra, Agung Pramana W.

    2018-01-01

    Autoimmune Thyroid Disease (AITD) is an autoimmune disease that has many clinical symptoms but is difficult to detect at the onset of disease progression. Most thyroid autoimmune disease patients are positive with high titre of thyroid autoantibodies, especially thyroid peroxidase (TPO). The detection AITD are still needed because these tests are extremely high cost and have not regularly been performed in most of clinical laboratories. In the past, we have explored the autoimmune disease marker and it has been developed as source of polyclonal antibodies from patient origin. In the current study, we develop recombinant protein which resulted from cloning and expression of TPO gene from normal person and AITD patients. This work flows involves: DNA isolation and PCR to obtain TPO gene from human blood, insertion of TPO gene to plasmid and transformation to E. coli BL21, Bacterial culture to obtain protein product, protein purification and product analysis. This products can use for application to immunochromatography based test. This work could achieved with the goal of producing autoimmune markers with a guaranteed quality, sensitive, specific and economically. So with the collaboration with industries these devices could be used for early detection. Keywords: recombinant protein, TPO gene, Autoimmune thyroid diseases (AITD)ction of the diseases in the community.

  18. Stable Plastid Transformation for High-Level Recombinant Protein Expression: Promises and Challenges

    Directory of Open Access Journals (Sweden)

    Meili Gao

    2012-01-01

    Full Text Available Plants are a promising expression system for the production of recombinant proteins. However, low protein productivity remains a major obstacle that limits extensive commercialization of whole plant and plant cell bioproduction platform. Plastid genetic engineering offers several advantages, including high levels of transgenic expression, transgenic containment via maternal inheritance, and multigene expression in a single transformation event. In recent years, the development of optimized expression strategies has given a huge boost to the exploitation of plastids in molecular farming. The driving forces behind the high expression level of plastid bioreactors include codon optimization, promoters and UTRs, genotypic modifications, endogenous enhancer and regulatory elements, posttranslational modification, and proteolysis. Exciting progress of the high expression level has been made with the plastid-based production of two particularly important classes of pharmaceuticals: vaccine antigens, therapeutic proteins, and antibiotics and enzymes. Approaches to overcome and solve the associated challenges of this culture system that include low transformation frequencies, the formation of inclusion bodies, and purification of recombinant proteins will also be discussed.

  19. Manufacturing recombinant proteins in kg-ton quantities using animal cells in bioreactors.

    Science.gov (United States)

    De Jesus, Maria; Wurm, Florian M

    2011-06-01

    Mammalian cells in bioreactors as production host are the focus of this review. We wish to briefly describe today's technical status and to highlight emerging trends in the manufacture of recombinant therapeutic proteins, focusing on Chinese hamster ovary (CHO) cells. CHO cells are the manufacturing host system of choice for more than 70% of protein pharmaceuticals on the market [21]. The current global capacity to grow mammalian cells in bioreactors stands at about 0.5 million liters, whereby the largest vessels can have a working volume of about 20,000l. We are focusing in this article on the upstream part of protein manufacturing. Over the past 25 years, volumetric yields for recombinant cell lines have increased about 20-fold mainly as the result of improvements in media and bioprocess design. Future yield increases are expected to come from improved gene delivery methods, from improved, possibly genetically modified host systems, and from further improved bioprocesses in bioreactors. Other emerging trends in protein manufacturing that are discussed include the use of disposal bioreactors and transient gene expression. We specifically highlight here current research in our own laboratories. Copyright © 2011 Elsevier B.V. All rights reserved.

  20. Protein misfolding cyclic amplification induces the conversion of recombinant prion protein to PrP oligomers causing neuronal apoptosis.

    Science.gov (United States)

    Yuan, Zhen; Yang, Lifeng; Chen, Baian; Zhu, Ting; Hassan, Mohammad Farooque; Yin, Xiaomin; Zhou, Xiangmei; Zhao, Deming

    2015-06-01

    The formation of neurotoxic prion protein (PrP) oligomers is thought to be a key step in the development of prion diseases. Recently, it was determined that the sonication and shaking of recombinant PrP can convert PrP monomers into β-state oligomers. Herein, we demonstrate that β-state oligomeric PrP can be generated through protein misfolding cyclic amplification from recombinant full-length hamster, human, rabbit, and mutated rabbit PrP, and that these oligomers can be used for subsequent research into the mechanisms of PrP-induced neurotoxicity. We have characterized protein misfolding cyclic amplification-induced monomer-to-oligomer conversion of PrP from three species using western blotting, circular dichroism, size-exclusion chromatography, and resistance to proteinase K (PK) digestion. We have further shown that all of the resulting β-oligomers are toxic to primary mouse cortical neurons independent of the presence of PrP(C) in the neurons, whereas the corresponding monomeric PrP were not toxic. In addition, we found that this toxicity is the result of oligomer-induced apoptosis via regulation of Bcl-2, Bax, and caspase-3 in both wild-type and PrP(-/-) cortical neurons. It is our hope that these results may contribute to our understanding of prion transformation within the brain. We found that β-state oligomeric PrPs can be generated through protein misfolding cyclic amplification (PMCA) from recombinant full-length hamster, human, rabbit, and mutated rabbit PrP. β-oligomers are toxic to primary mouse cortical neurons independent of the presence of PrP(C) in the neurons, while the corresponding monomeric PrPs were not toxic. This toxicity is the result of oligomers-induced apoptosis via regulation of Bcl-2, Bax, and caspase-3. These results may contribute to our understanding of prion transformation within the brain. © 2015 International Society for Neurochemistry.

  1. Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.

    Science.gov (United States)

    Wang, Hua-zhen; Chu, Zhi-zhan; Chen, Chang-chao; Cao, Ao-cheng; Tong, Xin; Ouyang, Can-bin; Yuan, Qi-hang; Wang, Mi-nan; Wu, Zhong-kun; Wang, Hai-hong; Wang, Sheng-bin

    2015-01-01

    Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.

  2. Importance of Heat and Pressure for Solubilization of Recombinant Spider Silk Proteins in Aqueous Solution.

    Science.gov (United States)

    Jones, Justin A; Harris, Thomas I; Oliveira, Paula F; Bell, Brianne E; Alhabib, Abdulrahman; Lewis, Randolph V

    2016-11-23

    The production of recombinant spider silk proteins continues to be a key area of interest for a number of research groups. Several key obstacles exist in their production as well as in their formulation into useable products. The original reported method to solubilize recombinant spider silk proteins (rSSp) in an aqueous solution involved using microwaves to quickly generate heat and pressure inside of a sealed vial containing rSSp and water. Fibers produced from this system are remarkable in their mechanical ability and demonstrate the ability to be stretched and recover 100 times. The microwave method dissolves the rSSPs with dissolution time increasing with higher molecular weight constructs, increasing concentration of rSSPs, protein type, and salt concentration. It has proven successful in solvating a number of different rSSPs including native-like sequences (MaSp1, MaSp2, piriform, and aggregate) as well as chimeric sequences (FlAS) in varied concentrations that have been spun into fibers and formed into films, foams, sponges, gels, coatings, macro and micro spheres and adhesives. The system is effective but inherently unpredictable and difficult to control. Provided that the materials that can be generated from this method of dissolution are impressive, an alternative means of applying heat and pressure that is controllable and predictable has been developed. Results indicate that there are combinations of heat and pressure (135 °C and 140 psi) that result in maximal dissolution without degrading the recombinant MaSp2 protein tested, and that heat and pressure are the key elements to the method of dissolution.

  3. Protein-only, antimicrobial peptide-containing recombinant nanoparticles with inherent built-in antibacterial activity.

    Science.gov (United States)

    Serna, Naroa; Sánchez-García, Laura; Sánchez-Chardi, Alejandro; Unzueta, Ugutz; Roldán, Mónica; Mangues, Ramón; Vázquez, Esther; Villaverde, Antonio

    2017-09-15

    The emergence of bacterial antibiotic resistances is a serious concern in human and animal health. In this context, naturally occurring cationic antimicrobial peptides (AMPs) might play a main role in a next generation of drugs against bacterial infections. Taking an innovative approach to design self-organizing functional proteins, we have generated here protein-only nanoparticles with intrinsic AMP microbicide activity. Using a recombinant version of the GWH1 antimicrobial peptide as building block, these materials show a wide antibacterial activity spectrum in absence of detectable toxicity on mammalian cells. The GWH1-based nanoparticles combine clinically appealing properties of nanoscale materials with full biocompatibility, structural and functional plasticity and biological efficacy exhibited by proteins. Because of the largely implemented biological fabrication of recombinant protein drugs, the protein-based platform presented here represents a novel and scalable strategy in antimicrobial drug design, that by solving some of the limitations of AMPs offers a promising alternative to conventional antibiotics. The low molecular weight antimicrobial peptide GWH1 has been engineered to oligomerize as self-assembling protein-only nanoparticles of around 50nm. In this form, the peptide exhibits potent and broad antibacterial activities against both Gram-positive and Gram-negative bacteria, without any harmful effect over mammalian cells. As a solid proof-of-concept, this finding strongly supports the design and biofabrication of nanoscale antimicrobial materials with in-built functionalities. The protein-based homogeneous composition offer advantages over alternative materials explored as antimicrobial agents, regarding biocompatibility, biodegradability and environmental suitability. Beyond the described prototype, this transversal engineering concept has wide applicability in the design of novel nanomedicines for advanced treatments of bacterial infections

  4. Genomic features shaping the landscape of meiotic double-strand-break hotspots in maize.

    Science.gov (United States)

    He, Yan; Wang, Minghui; Dukowic-Schulze, Stefanie; Zhou, Adele; Tiang, Choon-Lin; Shilo, Shay; Sidhu, Gaganpreet K; Eichten, Steven; Bradbury, Peter; Springer, Nathan M; Buckler, Edward S; Levy, Avraham A; Sun, Qi; Pillardy, Jaroslaw; Kianian, Penny M A; Kianian, Shahryar F; Chen, Changbin; Pawlowski, Wojciech P

    2017-11-14

    Meiotic recombination is the most important source of genetic variation in higher eukaryotes. It is initiated by formation of double-strand breaks (DSBs) in chromosomal DNA in early meiotic prophase. The DSBs are subsequently repaired, resulting in crossovers (COs) and noncrossovers (NCOs). Recombination events are not distributed evenly along chromosomes but cluster at recombination hotspots. How specific sites become hotspots is poorly understood. Studies in yeast and mammals linked initiation of meiotic recombination to active chromatin features present upstream from genes, such as absence of nucleosomes and presence of trimethylation of lysine 4 in histone H3 (H3K4me3). Core recombination components are conserved among eukaryotes, but it is unclear whether this conservation results in universal characteristics of recombination landscapes shared by a wide range of species. To address this question, we mapped meiotic DSBs in maize, a higher eukaryote with a large genome that is rich in repetitive DNA. We found DSBs in maize to be frequent in all chromosome regions, including sites lacking COs, such as centromeres and pericentromeric regions. Furthermore, most DSBs are formed in repetitive DNA, predominantly Gypsy retrotransposons, and only one-quarter of DSB hotspots are near genes. Genic and nongenic hotspots differ in several characteristics, and only genic DSBs contribute to crossover formation. Maize hotspots overlap regions of low nucleosome occupancy but show only limited association with H3K4me3 sites. Overall, maize DSB hotspots exhibit distribution patterns and characteristics not reported previously in other species. Understanding recombination patterns in maize will shed light on mechanisms affecting dynamics of the plant genome.

  5. Metabolic flux profiling of recombinant protein secreting Pichia pastoris growing on glucose:methanol mixtures

    Science.gov (United States)

    2012-01-01

    Background The methylotrophic yeast Pichia pastoris has emerged as one of the most promising yeast hosts for the production of heterologous proteins. Mixed feeds of methanol and a multicarbon source instead of methanol as sole carbon source have been shown to improve product productivities and alleviate metabolic burden derived from protein production. Nevertheless, systematic quantitative studies on the relationships between the central metabolism and recombinant protein production in P. pastoris are still rather limited, particularly when growing this yeast on mixed carbon sources, thus hampering future metabolic network engineering strategies for improved protein production. Results The metabolic flux distribution in the central metabolism of P. pastoris growing on a mixed feed of glucose and methanol was analyzed by Metabolic Flux Analysis (MFA) using 13C-NMR-derived constraints. For this purpose, we defined new flux ratios for methanol assimilation pathways in P. pastoris cells growing on glucose:methanol mixtures. By using this experimental approach, the metabolic burden caused by the overexpression and secretion of a Rhizopus oryzae lipase (Rol) in P. pastoris was further analyzed. This protein has been previously shown to trigger the unfolded protein response in P. pastoris. A series of 13C-tracer experiments were performed on aerobic chemostat cultivations with a control and two different Rol producing strains growing at a dilution rate of 0.09 h−1 using a glucose:methanol 80:20 (w/w) mix as carbon source. The MFA performed in this study reveals a significant redistristribution of carbon fluxes in the central carbon metabolism when comparing the two recombinant strains vs the control strain, reflected in increased glycolytic, TCA cycle and NADH regeneration fluxes, as well as higher methanol dissimilation rates. Conclusions Overall, a further 13C-based MFA development to characterise the central metabolism of methylotrophic yeasts when growing on mixed

  6. Metabolic flux profiling of recombinant protein secreting Pichia pastoris growing on glucose:methanol mixtures

    Directory of Open Access Journals (Sweden)

    Jordà Joel

    2012-05-01

    Full Text Available Abstract Background The methylotrophic yeast Pichia pastoris has emerged as one of the most promising yeast hosts for the production of heterologous proteins. Mixed feeds of methanol and a multicarbon source instead of methanol as sole carbon source have been shown to improve product productivities and alleviate metabolic burden derived from protein production. Nevertheless, systematic quantitative studies on the relationships between the central metabolism and recombinant protein production in P. pastoris are still rather limited, particularly when growing this yeast on mixed carbon sources, thus hampering future metabolic network engineering strategies for improved protein production. Results The metabolic flux distribution in the central metabolism of P. pastoris growing on a mixed feed of glucose and methanol was analyzed by Metabolic Flux Analysis (MFA using 13C-NMR-derived constraints. For this purpose, we defined new flux ratios for methanol assimilation pathways in P. pastoris cells growing on glucose:methanol mixtures. By using this experimental approach, the metabolic burden caused by the overexpression and secretion of a Rhizopus oryzae lipase (Rol in P. pastoris was further analyzed. This protein has been previously shown to trigger the unfolded protein response in P. pastoris. A series of 13C-tracer experiments were performed on aerobic chemostat cultivations with a control and two different Rol producing strains growing at a dilution rate of 0.09 h−1 using a glucose:methanol 80:20 (w/w mix as carbon source. The MFA performed in this study reveals a significant redistristribution of carbon fluxes in the central carbon metabolism when comparing the two recombinant strains vs the control strain, reflected in increased glycolytic, TCA cycle and NADH regeneration fluxes, as well as higher methanol dissimilation rates. Conclusions Overall, a further 13C-based MFA development to characterise the central metabolism of methylotrophic

  7. Preparation and use of recombinant protein G-gold complexes as markers in double labelling immunocytochemistry

    DEFF Research Database (Denmark)

    Balslev, Y; Hansen, Gert Helge

    1989-01-01

    Recombinant protein G (RPG) was conjugated to colloidal gold particles and used for immunocytochemistry. In this report, the preparation of RPG-gold conjugates (RPGG) and the application of these conjugates in spot blot tests and in double immunolabelling are described. The immunolabelling...... was performed on ultracryosections of pig small intestine using antibodies directed against aminopeptidase N and sucrase-isomaltase. The labelling efficiency of RPGG was compared to that of protein A-gold conjugates (PAG) in different compartments of the enterocyte. Quantification showed that the labelling...... intensity was dependent on the size of the marker as well as on the kind of protein used for complex formation. The distributions for RPGG and PAG were respectively: for the 12 nm particles, 10.3 and 6.2 particles/micron of length of microvillar membrane, 3.5 and 1.0 particles/micron2 of Golgi profile and 5...

  8. Serological Analysis of Immunogenic Properties of Recombinant Meningococcus IgA1 Protease-Based Proteins.

    Science.gov (United States)

    Kotelnikova, O V; Zinchenko, A A; Vikhrov, A A; Alliluev, A P; Serova, O V; Gordeeva, E A; Zhigis, L S; Zueva, V S; Razgulyaeva, O A; Melikhova, T D; Nokel, E A; Drozhzhina, E Yu; Rumsh, L D

    2016-07-01

    Using the genome sequence of IgA1 protease of N. meningitidis of serogroup B, four recombinant proteins of different structure and molecular weight were constructed. These proteins were equal in inducing the formation of specific antibodies to IgA1 protease and had protective properties against meningococci. In the sera of immunized mice, anti-IgA1 protease antibodies were detected by whole-cell ELISA, which indicated the presence of IgA1 protease on the surface of these bacteria. We hypothesized that the protective properties of IgA1 protease-based antigens and IgA1 protease analogs could be realized not only via impairment of bacterium adhesion to the mucosa, but also via suppression of this pathogen in the organism. The presented findings seem promising for using these proteins as the basis for anti-meningococcus vaccine.

  9. Extensive Lysine Methylation in Hyperthermophilic Crenarchaea: Potential Implications for Protein Stability and Recombinant Enzymes

    Directory of Open Access Journals (Sweden)

    Catherine H. Botting

    2010-01-01

    Full Text Available In eukarya and bacteria, lysine methylation is relatively rare and is catalysed by sequence-specific lysine methyltransferases that typically have only a single-protein target. Using RNA polymerase purified from the thermophilic crenarchaeum Sulfolobus solfataricus, we identified 21 methyllysines distributed across 9 subunits of the enzyme. The modified lysines were predominantly in α-helices and showed no conserved sequence context. A limited survey of the Thermoproteus tenax proteome revealed widespread modification with 52 methyllysines in 30 different proteins. These observations suggest the presence of an unusual lysine methyltransferase with relaxed specificity in the crenarchaea. Since lysine methylation is known to enhance protein thermostability, this may be an adaptation to a thermophilic lifestyle. The implications of this modification for studies and applications of recombinant crenarchaeal enzymes are discussed.

  10. Immunogenicity of recombinant class 1 protein from Neisseria meningitidis refolded into phospholipid vesicles and detergent.

    Science.gov (United States)

    Niebla, O; Alvarez, A; Martín, A; Rodríguez, A; Delgado, M; Falcón, V; Guillén, G

    2001-05-14

    The possibility of eliciting bactericidal antibodies against a recombinant class 1 protein (P1) from Neisseria meningitidis, joined to the first 45 amino acids of the neisserial LpdA protein (PM82), was examined. P1 was produced in Escherichia coli as intracellular inclusion bodies, from which it was purified and reconstituted by (a) inclusion into phospholipid vesicles and detergent and (b) refolding in 0.1% SDS. When Balb/c mice were immunised, high titres of subtype-specific bactericidal antibodies against P1 were obtained in both cases. These results suggest that in spite of being a denaturing agent, it is possible to use SDS to reconstitute the P1 protein in a conformation that exposes the immunodominat regions.

  11. Protective efficacy of six immunogenic recombinant proteins of Vibrio anguillarum and evaluation them as vaccine candidate for flounder (Paralichthys olivaceus).

    Science.gov (United States)

    Xing, Jing; Xu, Hongsen; Wang, Yang; Tang, Xiaoqian; Sheng, Xiuzhen; Zhan, Wenbin

    2017-06-01

    Vibrio anguillarum is a severe bacterium that causes terminal haemorrhagic septicaemia in freshwater and marine fish. Virulence-associated proteins play an important role in bacterial pathogenicity and could be applied for immunoprophylaxis. In this study, six antigenic proteins from V. anguillarum were selected and the immune protective efficacy of their recombinant proteins was investigated. VirA, CheR, FlaC, OmpK, OmpR and Hsp33 were recombinantly produced and the reactions of recombinant proteins to flounder-anti-V. anguillarum antibodies (fV-ab) were detected, respectively. Then the recombinant proteins were injected to fish, after immunization, the percentages of surface membrane immunoglobulin-positive (sIg+) cell in lymphocytes, total antibodies, antibodies against V. anguillarum, antibodies against recombinant proteins and relative percent survival (RPS) were analyzed, respectively. The results showed that all the recombinant proteins could react to fV-ab, proliferate sIg + cells in lymphocytes and induce production of total antibodies, specific antibodies against V. anguillarum or the recombinant proteins; the RPS of rVirA, rCheR, rFlaC, rOmpK, rOmpR and rHsp33 against V. anguillarum was 70.27%, 27.03%, 16.22%, 62.16%, 45.95% and 81.08%, respectively. The results revealed that rHsp33, rVirA and rOmpK have good protections against V. anguillarum and could be vaccine candidates against V. anguillarum. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. The recombinant globular head domain of the measles virus hemagglutinin protein as a subunit vaccine against measles.

    Science.gov (United States)

    Lobanova, Liubov M; Eng, Nelson F; Satkunarajah, Malathy; Mutwiri, George K; Rini, James M; Zakhartchouk, Alexander N

    2012-04-26

    Despite the availability of live attenuated measles virus (MV) vaccines, a large number of measles-associated deaths occur among infants in developing countries. The development of a measles subunit vaccine may circumvent the limitations associated with the current live attenuated vaccines and eventually contribute to global measles eradication. Therefore, the goal of this study was to test the feasibility of producing the recombinant globular head domain of the MV hemagglutinin (H) protein by stably transfected human cells and to examine the ability of this recombinant protein to elicit MV-specific immune responses. The recombinant protein was purified from the culture supernatant of stably transfected HEK293T cells secreting a tagged version of the protein. Two subcutaneous immunizations with the purified recombinant protein alone resulted in the production of MV-specific serum IgG and neutralizing antibodies in mice. Formulation of the protein with adjuvants (polyphosphazene or alum) further enhanced the humoral immune response and in addition resulted in the induction of cell-mediated immunity as measured by the production of MV H-specific interferon gamma (IFN-γ) and interleukin 5 (IL-5) by in vitro re-stimulated splenocytes. Furthermore, the inclusion of polyphosphazene into the vaccine formulation induced a mixed Th1/Th2-type immune response. In addition, the purified recombinant protein retained its immunogenicity even after storage at 37°C for 2 weeks. Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. Prdm9 Controls Activation of Mammalian Recombination Hotspots

    OpenAIRE

    Parvanov, Emil D.; Petkov, Petko M.; Paigen, Kenneth

    2009-01-01

    Mammalian meiotic recombination, which preferentially occurs at specialized sites called hotspots, assures the orderly segregation of meiotic chromosomes and creates genetic variation among offspring. A locus on mouse Chr 17, that controls activation of recombination at multiple distant hotspots, has been mapped within a 181 Kb interval, three of whose genes can be eliminated as candidates. The remaining gene, Prdm9, codes for a zinc finger containing histone H3K4 trimethylase that is uniquel...

  14. Cell culture media supplementation of uncommonly used sugars sucrose and tagatose for the targeted shifting of protein glycosylation profiles of recombinant protein therapeutics.

    Science.gov (United States)

    Hossler, Patrick; McDermott, Sean; Racicot, Christopher; Chumsae, Christopher; Raharimampionona, Haly; Zhou, Yu; Ouellette, David; Matuck, Joseph; Correia, Ivan; Fann, John; Li, Jianmin

    2014-01-01

    Protein glycosylation is an important post-translational modification toward the structure and function of recombinant therapeutics. The addition of oligosaccharides to recombinant proteins has been shown to greatly influence the overall physiochemical attributes of many proteins. It is for this reason that protein glycosylation is monitored by the developer of a recombinant protein therapeutic, and why protein glycosylation is typically considered a critical quality attribute. In this work, we highlight a systematic study toward the supplementation of sucrose and tagatose into cell culture media for the targeted modulation of protein glycosylation profiles on recombinant proteins. Both sugars were found to affect oligosaccharide maturation resulting in an increase in the percentage of high mannose N-glycan species, as well as a concomitant reduction in fucosylation. The latter effect was demonstrated to increase antibody-dependent cell-mediated cytotoxicity for a recombinant antibody. These aforementioned results were found to be reproducible at different scales, and across different Chinese hamster ovary cell lines. Through the selective supplementation of these described sugars, the targeted modulation of protein glycosylation profiles is demonstrated, as well as yet another tool in the cell culture toolbox for ensuring product comparability. © 2014 American Institute of Chemical Engineers.

  15. A recombinant pseudorabies virus co-expressing capsid proteins precursor P1-2A of FMDV and VP2 protein of porcine parvovirus: a trivalent vaccine candidate.

    Science.gov (United States)

    Hong, Qi; Qian, Ping; Li, Xiang-Min; Yu, Xiao-Lan; Chen, Huan-Chun

    2007-11-01

    Pseudorabies (PR), foot-and-mouth disease (FMD), and porcine parvovirus disease are three important infectious diseases in swine worldwide. The gene-deleted pseudorabies virus (PRV) has been used as a live-viral vector to develop multivalent genetic engineering vaccine. In this study, a recombinant PRV, which could co-express protein precursor P1-2A of FMDV and VP2 protein of PPV, was constructed using PRV TK(-)/gE(-)/LacZ(+) mutant as the vector. After homologous recombination and plaque purification, recombinant virus PRV TK(-)/gE(-)/P1-2A-VP2 was acquired and identified. Immunogenicity, safety of the recombinant PRV and its protection against PRV were confirmed in a mouse model by indirect ELISA and serum neutralization test. The results show that the recombinant PRV is a candidate vaccine strain to develop a novel trivalent vaccine against PRV, FMDV and PPV in swine.

  16. Deleting multiple lytic genes enhances biomass yield and production of recombinant proteins by Bacillus subtilis.

    Science.gov (United States)

    Wang, Yi; Chen, Zhenmin; Zhao, Ruili; Jin, Tingting; Zhang, Xiaoming; Chen, Xiangdong

    2014-08-31

    Bacillus subtilis is widely used in agriculture and industrial biotechnology; however, cell autolysis significantly decreases its yield in liquid cultures. Numerous factors mediate the lysis of B. subtilis, such as cannibalism factors, prophages, and peptidoglycan (PG) hydrolases. The aim of this work was to use molecular genetic techniques to develop a new strategy to prevent cell lysis and enhance biomass as well as the production of recombinant proteins. Five genes or genetic elements representing three different functional categories were studied as follows: lytC encoding PG hydrolases, the prophage genes xpf and yqxG-yqxH-cwlA (yGlA), and skfA and sdpC that encode cannibalism factors. Cell lysis was reduced and biomass was enhanced by deleting individually skfA, sdpC, xpf, and lytC. We constructed the multiple deletion mutant LM2531 (skfA sdpC lytC xpf) and found that after 4 h of culture, its biomass yield was significantly increased compared with that of prototypical B. subtilis 168 (wild-type) strain and that 15% and 92% of the cells were lysed in cultures of LM2531 and wild-type, respectively. Moreover, two expression vectors were constructed for producing recombinant proteins (β-galactosidase and nattokinase) under the control of the P43 promoter. Cultures of LM2531 and wild-type transformants produced 13741 U/ml and 7991 U/ml of intracellular β-galactosidase, respectively (1.72-fold increase). Further, the level of secreted nattokinase produced by strain LM2531 increased by 2.6-fold compared with wild-type (5226 IU/ml vs. 2028 IU/ml, respectively). Our novel, systematic multigene deletion approach designed to inhibit cell lysis significantly increased the biomass yield and the production of recombinant proteins by B. subtilis. These findings show promise for guiding efforts to manipulate the genomes of other B. subtilis strains that are used for industrial purposes.

  17. Recombinant VSV G proteins reveal a novel raft-dependent endocytic pathway in resorbing osteoclasts

    International Nuclear Information System (INIS)

    Mulari, Mika T.K.; Nars, Martin; Laitala-Leinonen, Tiina; Kaisto, Tuula; Metsikkoe, Kalervo; Sun Yi; Vaeaenaenen, H. Kalervo

    2008-01-01

    Transcytotic membrane flow delivers degraded bone fragments from the ruffled border to the functional secretory domain, FSD, in bone resorbing osteoclasts. Here we show that there is also a FSD-to-ruffled border trafficking pathway that compensates for the membrane loss during the matrix uptake process and that rafts are essential for this ruffled border-targeted endosomal pathway. Replacing the cytoplasmic tail of the vesicular stomatitis virus G protein with that of CD4 resulted in partial insolubility in Triton X-100 and retargeting from the peripheral non-bone facing plasma membrane to the FSD. Recombinant G proteins were subsequently endosytosed and delivered from the FSD to the peripheral fusion zone of the ruffled border, which were both rich in lipid rafts as suggested by viral protein transport analysis and visualizing the rafts with fluorescent recombinant cholera toxin. Cholesterol depletion by methyl-β-cyclodextrin impaired the ruffled border-targeted vesicle trafficking pathway and inhibited bone resorption dose-dependently as quantified by measuring the CTX and TRACP 5b secreted to the culture medium and by measuring the resorbed area visualized with a bi-phasic labeling method using sulpho-NHS-biotin and WGA-lectin. Thus, rafts are vital for membrane recycling from the FSD to the late endosomal/lysosomal ruffled border and bone resorption

  18. The evolutionary turnover of recombination hot spots contributes to speciation in mice.

    Science.gov (United States)

    Smagulova, Fatima; Brick, Kevin; Pu, Yongmei; Camerini-Otero, R Daniel; Petukhova, Galina V

    2016-02-01

    Meiotic recombination is required for the segregation of homologous chromosomes and is essential for fertility. In most mammals, the DNA double-strand breaks (DSBs) that initiate meiotic recombination are directed to a subset of genomic loci (hot spots) by sequence-specific binding of the PRDM9 protein. Rapid evolution of the DNA-binding specificity of PRDM9 and gradual erosion of PRDM9-binding sites by gene conversion will alter the recombination landscape over time. To better understand the evolutionary turnover of recombination hot spots and its consequences, we mapped DSB hot spots in four major subspecies of Mus musculus with different Prdm9 alleles and in their F1 hybrids. We found that hot spot erosion governs the preferential usage of some Prdm9 alleles over others in hybrid mice and increases sequence diversity specifically at hot spots that become active in the hybrids. As crossovers are disfavored at such hot spots, we propose that sequence divergence generated by hot spot turnover may create an impediment for recombination in hybrids, potentially leading to reduced fertility and, eventually, speciation. Published by Cold Spring Harbor Laboratory Press.

  19. Positive and negative regulation of V(D)J recombination by the E2A proteins.

    Science.gov (United States)

    Bain, G; Romanow, W J; Albers, K; Havran, W L; Murre, C

    1999-01-18

    A key feature of B and T lymphocyte development is the generation of antigen receptors through the rearrangement and assembly of the germline variable (V), diversity (D), and joining (J) gene segments. However, the mechanisms responsible for regulating developmentally ordered gene rearrangements are largely unknown. Here we show that the E2A gene products are essential for the proper coordinated temporal regulation of V(D)J rearrangements within the T cell receptor (TCR) gamma and delta loci. Specifically, we show that E2A is required during adult thymocyte development to inhibit rearrangements to the gamma and delta V regions that normally recombine almost exclusively during fetal thymocyte development. The continued rearrangement of the fetal Vgamma3 gene segment in E2A-deficient adult thymocytes correlates with increased levels of Vgamma3 germline transcripts and increased levels of double-stranded DNA breaks at the recombination signal sequence bordering Vgamma3. Additionally, rearrangements to a number of Vgamma and Vdelta gene segments used predominantly during adult development are significantly reduced in E2A-deficient thymocytes. Interestingly, at distinct stages of T lineage development, both the increased and decreased rearrangement of particular Vdelta gene segments is highly sensitive to the dosage of the E2A gene products, suggesting that the concentration of the E2A proteins is rate limiting for the recombination reaction involving these Vdelta regions.

  20. Diagnostic value of recombinant Tp0821 protein in serodiagnosis for syphilis.

    Science.gov (United States)

    Xie, Y; Xu, M; Wang, C; Xiao, J; Xiao, Y; Jiang, C; You, X; Zhao, F; Zeng, T; Liu, S; Kuang, X; Wu, Y

    2016-04-01

    Syphilis is a multistage sexually transmitted disease that remains a serious public health concern worldwide. The coexistence of Treponema pallidum with other closely related members of spirochaeta, such as Leptospira spp. and Borrelia burgdorferi, has complicated the serodiagnosis due to cross-reactive antigens. In this study, recombinant Tp0821 protein was expressed in Escherichia coli and purified by metal affinity chromatography. Then enzyme-linked immunosorbent assays (ELISAs) based on Tp0821 for the detection of specific antibodies were established. The relative positive rates of the IgM ELISA and the IgG ELISA were found to be 91·0 and 98·3%, respectively, when screening 578 syphilis specimens. The specificities were 94·3 and 100%, respectively, when cross-checking with serum samples obtained from 30 patients with Lyme disease, five patients with leptospirosis, and 52 uninfected controls. In addition, relative positive rates and specificities of Tp0821 for human sera were all 100% in Western blotting. When compared to the syphilis diagnostic tests commonly used in clinical settings, we found that the results of Tp0821-based ELISAs correlated well with the results of the treponemal tests, specifically the T. pallidum particle agglutination (TP-PA) test and the chemiluminescent immunoassay (CIA). Thus, these findings identify Tp0821 as a novel serodiagnostic candidate for syphilis. In this study, we expressed and purified the Treponema pallidum protein Tp0821 and developed Tp0821-based enzyme-linked immunosorbent assays (ELISAs) for the detection of specific antibodies. The serodiagnostic performance of the recombinant protein was then evaluated. When compared to the results of syphilis diagnostic tests commonly used in clinical settings, we found that the reactivities of syphilitic sera with the recombinant antigen correlated well with the results of the treponemal tests, specifically the T. pallidum particle agglutination (TP-PA) test and the

  1. Maxillary anterior ridge augmentation with recombinant human bone morphogenetic protein 2.

    Science.gov (United States)

    Edmunds, Ryan K; Mealey, Brian L; Mills, Michael P; Thoma, Daniel S; Schoolfield, John; Cochran, David L; Mellonig, Jim

    2014-01-01

    No human studies exist on the use of recombinant human bone morphogenetic protein 2 (rhBMP-2) on an absorbable collagen sponge (ACS) as a sole graft material for lateral ridge augmentation in large ridge defect sites. This series evaluates the treatment outcome of maxillary anterior lateral ridge augmentation with rhBMP-2/ACS. Twenty patients were treated with rhBMP-2/ACS and fixation screws for space maintenance. Cone beam volumetric tomography measurements were used to determine gain in ridge width, and a bone core biopsy was obtained. The mean horizontal ridge gain was 1.2 mm across sites, and every site gained width.

  2. Effective non-denaturing purification method for improving the solubility of recombinant actin-binding proteins produced by bacterial expression.

    Science.gov (United States)

    Chung, Jeong Min; Lee, Sangmin; Jung, Hyun Suk

    2017-05-01

    Bacterial expression is commonly used to produce recombinant and truncated mutant eukaryotic proteins. However, heterologous protein expression may render synthesized proteins insoluble. The conventional method used to express a poorly soluble protein, which involves denaturation and refolding, is time-consuming and inefficient. There are several non-denaturing approaches that can increase the solubility of recombinant proteins that include using different bacterial cell strains, altering the time of induction, lowering the incubation temperature, and employing different detergents for purification. In this study, we compared several non-denaturing protocols to express and purify two insoluble 34 kDa actin-bundling protein mutants. The solubility of the mutant proteins was not affected by any of the approaches except for treatment with the detergent sarkosyl. These results indicate that sarkosyl can effectively improve the solubility of insoluble proteins during bacterial expression. Copyright © 2016. Published by Elsevier Inc.

  3. Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway

    Science.gov (United States)

    2012-01-01

    Βackground The methylotrophic yeast Pichia pastoris has become an important host organism for recombinant protein production and is able to use methanol as a sole carbon source. The methanol utilization pathway describes all the catalytic reactions, which happen during methanol metabolism. Despite the importance of certain key enzymes in this pathway, so far very little is known about possible effects of overexpressing either of these key enzymes on the overall energetic behavior, the productivity and the substrate uptake rate in P. pastoris strains. Results A fast and easy-to-do approach based on batch cultivations with methanol pulses was used to characterize different P. pastoris strains. A strain with MutS phenotype was found to be superior over a strain with Mut+ phenotype in both the volumetric productivity and the efficiency in expressing recombinant horseradish peroxidase C1A. Consequently, either of the enzymes dihydroxyacetone synthase, transketolase or formaldehyde dehydrogenase, which play key roles in the methanol utilization pathway, was co-overexpressed in MutS strains harboring either of the reporter enzymes horseradish peroxidase or Candida antarctica lipase B. Although the co-overexpression of these enzymes did not change the stoichiometric yields of the recombinant MutS strains, significant changes in the specific growth rate, the specific substrate uptake rate and the specific productivity were observed. Co-overexpression of dihydroxyacetone synthase yielded a 2- to 3-fold more efficient conversion of the substrate methanol into product, but also resulted in a reduced volumetric productivity. Co-overexpression of formaldehyde dehydrogenase resulted in a 2-fold more efficient conversion of the substrate into product and at least similar volumetric productivities compared to strains without an engineered methanol utilization pathway, and thus turned out to be a valuable strategy to improve recombinant protein production. Conclusions Co

  4. Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway

    Directory of Open Access Journals (Sweden)

    Krainer Florian W

    2012-02-01

    Full Text Available Abstract Βackground The methylotrophic yeast Pichia pastoris has become an important host organism for recombinant protein production and is able to use methanol as a sole carbon source. The methanol utilization pathway describes all the catalytic reactions, which happen during methanol metabolism. Despite the importance of certain key enzymes in this pathway, so far very little is known about possible effects of overexpressing either of these key enzymes on the overall energetic behavior, the productivity and the substrate uptake rate in P. pastoris strains. Results A fast and easy-to-do approach based on batch cultivations with methanol pulses was used to characterize different P. pastoris strains. A strain with MutS phenotype was found to be superior over a strain with Mut+ phenotype in both the volumetric productivity and the efficiency in expressing recombinant horseradish peroxidase C1A. Consequently, either of the enzymes dihydroxyacetone synthase, transketolase or formaldehyde dehydrogenase, which play key roles in the methanol utilization pathway, was co-overexpressed in MutS strains harboring either of the reporter enzymes horseradish peroxidase or Candida antarctica lipase B. Although the co-overexpression of these enzymes did not change the stoichiometric yields of the recombinant MutS strains, significant changes in the specific growth rate, the specific substrate uptake rate and the specific productivity were observed. Co-overexpression of dihydroxyacetone synthase yielded a 2- to 3-fold more efficient conversion of the substrate methanol into product, but also resulted in a reduced volumetric productivity. Co-overexpression of formaldehyde dehydrogenase resulted in a 2-fold more efficient conversion of the substrate into product and at least similar volumetric productivities compared to strains without an engineered methanol utilization pathway, and thus turned out to be a valuable strategy to improve recombinant protein

  5. Structural and functional adaptations of the mammalian nuclear envelope to meet the meiotic requirements.

    Science.gov (United States)

    Link, Jana; Jahn, Daniel; Alsheimer, Manfred

    2015-01-01

    Numerous studies in the past years provided definite evidence that the nuclear envelope is much more than just a simple barrier. It rather constitutes a multifunctional platform combining structural and dynamic features to fulfill many fundamental functions such as chromatin organization, regulation of transcription, signaling, but also structural duties like maintaining general nuclear architecture and shape. One additional and, without doubt, highly impressive aspect is the recently identified key function of selected nuclear envelope components in driving meiotic chromosome dynamics, which in turn is essential for accurate recombination and segregation of the homologous chromosomes. Here, we summarize the recent work identifying new key players in meiotic telomere attachment and movement and discuss the latest advances in our understanding of the actual function of the meiotic nuclear envelope.

  6. Homologous recombination in mammalian cells: effect of p53 and Bcl-2 proteins, replication inhibition and ionizing radiations

    International Nuclear Information System (INIS)

    Saintigny, Yannick

    1999-01-01

    The control of cell cycle, associated with the mechanisms of replication, DNA repair/recombination allows the cells to maintain their genetic integrity. The p53 protein ensures the control of G1/S transition. Its inactivation would allow to initial replication on damaged matrix and lead to the block of replication forks followed by DNA strand breaks, good substrates for recombination. This work shows that the expression of mutant p53 protein stimulates both spontaneous and radio-induced homologous recombination, independently of the control of cell cycle. Moreover, the use of a set of replication inhibitors show that inhibition of the replication elongation stimulates recombination more strongly than the initiation inhibition. Replication arrest by these inhibitors also significantly increases the number of DNA strand breaks. These results highlighted a point of action of p53 protein on the ultimate stages of the homologous recombination mechanism. Lastly, the expression of Bcl-2 protein inhibits apoptosis and increases survival, but specifically inhibits conservative recombination, after radiation as well as in absence of apoptotic stress. The extinction of this mechanism of DNA repair is associated with an increase of mutagenesis. Taken together, these results allow ta consider the maintenance of the genetic stability as a cellular network involving different pathways. A multiple stages model for tumoral progression can be deduced. (author) [fr

  7. Recombinant production and solution structure of lipid transfer protein from lentil Lens culinaris

    Energy Technology Data Exchange (ETDEWEB)

    Gizatullina, Albina K. [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya str., 16/10, 117997 Moscow (Russian Federation); Moscow Institute of Physics and Technology (State University), Department of Physicochemical Biology and Biotechnology, Institutskii per., 9, 141700, Dolgoprudny, Moscow Region (Russian Federation); Finkina, Ekaterina I.; Mineev, Konstantin S.; Melnikova, Daria N.; Bogdanov, Ivan V. [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya str., 16/10, 117997 Moscow (Russian Federation); Telezhinskaya, Irina N.; Balandin, Sergey V. [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya str., 16/10, 117997 Moscow (Russian Federation); Moscow Institute of Physics and Technology (State University), Department of Physicochemical Biology and Biotechnology, Institutskii per., 9, 141700, Dolgoprudny, Moscow Region (Russian Federation); Shenkarev, Zakhar O. [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya str., 16/10, 117997 Moscow (Russian Federation); Arseniev, Alexander S. [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya str., 16/10, 117997 Moscow (Russian Federation); Moscow Institute of Physics and Technology (State University), Department of Physicochemical Biology and Biotechnology, Institutskii per., 9, 141700, Dolgoprudny, Moscow Region (Russian Federation); Ovchinnikova, Tatiana V., E-mail: ovch@ibch.ru [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya str., 16/10, 117997 Moscow (Russian Federation); Moscow Institute of Physics and Technology (State University), Department of Physicochemical Biology and Biotechnology, Institutskii per., 9, 141700, Dolgoprudny, Moscow Region (Russian Federation)

    2013-10-04

    Highlights: •Lipid transfer protein from lentil seeds (Lc-LTP2) was overexpressed in E. coli. •Antimicrobial activity and spatial structure of the recombinant Lc-LTP2 were examined. •Internal tunnel-like lipid-binding cavity occupies ∼7% of the total Lc-LTP2 volume. •Binding of DMPG lipid induces moderate rearrangements in the Lc-LTP2 structure. •Lc-LTP2/DMPG complex has limited lifetime and dissociates within tens of hours. -- Abstract: Lipid transfer protein, designated as Lc-LTP2, was isolated from seeds of the lentil Lens culinaris. The protein has molecular mass 9282.7 Da, consists of 93 amino acid residues including 8 cysteines forming 4 disulfide bonds. Lc-LTP2 and its stable isotope labeled analogues were overexpressed in Escherichia coli and purified. Antimicrobial activity of the recombinant protein was examined, and its spatial structure was studied by NMR spectroscopy. The polypeptide chain of Lc-LTP2 forms four α-helices (Cys4-Leu18, Pro26-Ala37, Thr42-Ala56, Thr64-Lys73) and a long C-terminal tail without regular secondary structure. Side chains of the hydrophobic residues form a relatively large internal tunnel-like lipid-binding cavity (van der Waals volume comes up to ∼600 Å{sup 3}). The side-chains of Arg45, Pro79, and Tyr80 are located near an assumed mouth of the cavity. Titration with dimyristoyl phosphatidylglycerol (DMPG) revealed formation of the Lc-LTP2/lipid non-covalent complex accompanied by rearrangements in the protein spatial structure and expansion of the internal cavity. The resultant Lc-LTP2/DMPG complex demonstrates limited lifetime and dissociates within tens of hours.

  8. Recombinant production and solution structure of lipid transfer protein from lentil Lens culinaris

    International Nuclear Information System (INIS)

    Gizatullina, Albina K.; Finkina, Ekaterina I.; Mineev, Konstantin S.; Melnikova, Daria N.; Bogdanov, Ivan V.; Telezhinskaya, Irina N.; Balandin, Sergey V.; Shenkarev, Zakhar O.; Arseniev, Alexander S.; Ovchinnikova, Tatiana V.

    2013-01-01

    Highlights: •Lipid transfer protein from lentil seeds (Lc-LTP2) was overexpressed in E. coli. •Antimicrobial activity and spatial structure of the recombinant Lc-LTP2 were examined. •Internal tunnel-like lipid-binding cavity occupies ∼7% of the total Lc-LTP2 volume. •Binding of DMPG lipid induces moderate rearrangements in the Lc-LTP2 structure. •Lc-LTP2/DMPG complex has limited lifetime and dissociates within tens of hours. -- Abstract: Lipid transfer protein, designated as Lc-LTP2, was isolated from seeds of the lentil Lens culinaris. The protein has molecular mass 9282.7 Da, consists of 93 amino acid residues including 8 cysteines forming 4 disulfide bonds. Lc-LTP2 and its stable isotope labeled analogues were overexpressed in Escherichia coli and purified. Antimicrobial activity of the recombinant protein was examined, and its spatial structure was studied by NMR spectroscopy. The polypeptide chain of Lc-LTP2 forms four α-helices (Cys4-Leu18, Pro26-Ala37, Thr42-Ala56, Thr64-Lys73) and a long C-terminal tail without regular secondary structure. Side chains of the hydrophobic residues form a relatively large internal tunnel-like lipid-binding cavity (van der Waals volume comes up to ∼600 Å 3 ). The side-chains of Arg45, Pro79, and Tyr80 are located near an assumed mouth of the cavity. Titration with dimyristoyl phosphatidylglycerol (DMPG) revealed formation of the Lc-LTP2/lipid non-covalent complex accompanied by rearrangements in the protein spatial structure and expansion of the internal cavity. The resultant Lc-LTP2/DMPG complex demonstrates limited lifetime and dissociates within tens of hours

  9. Factors Affecting the Binding of a Recombinant Heavy Metal-Binding Domain (CXXC motif Protein to Heavy Metals

    Directory of Open Access Journals (Sweden)

    Kamala Boonyodying

    2012-06-01

    Full Text Available A number of heavy metal-binding proteins have been used to study bioremediation. CXXC motif, a metal binding domain containing Cys-X-X-Cys motif, has been identified in various organisms. These proteins are capable of binding various types of heavy metals. In this study, heavy metal binding domain (CXXC motif recombinant protein encoded from mcsA gene of S. aureus were cloned and overexpressed in Escherichia coli. The factors involved in the metal-binding activity were determined in order to analyze the potential of recombinant protein for bioremediation. A recombinant protein can be bound to Cd2+, Co2+, Cu2+ and Zn2+. The thermal stability of a recombinant protein was tested, and the results showed that the metal binding activity to Cu2+ and Zn2+ still exist after treating the protein at 85ºC for 30 min. The temperature and pH that affected the metal binding activity was tested and the results showed that recombinant protein was still bound to Cu2+ at 65ºC, whereas a pH of 3-7 did not affect the metal binding E. coli harboring a pRset with a heavy metal-binding domain CXXC motif increased the resistance of heavy metals against CuCl2 and CdCl2. This study shows that metal binding domain (CXXC motif recombinant protein can be effectively bound to various types of heavy metals and may be used as a potential tool for studying bioremediation.

  10. Relationship between recombinant protein expression and host metabolome as determined by two-dimensional NMR spectroscopy.

    Directory of Open Access Journals (Sweden)

    Young Kee Chae

    Full Text Available Escherichia coli has been the most widely used host to produce large amounts of heterologous proteins. However, given an input plasmid DNA, E. coli may produce soluble protein, produce only inclusion bodies, or yield little or no protein at all. Many efforts have been made to surmount these problems, but most of them have involved time-consuming and labor-intensive trial-and-error. We hypothesized that different metabolomic fingerprints might be associated with different protein production outcomes. If so, then it might be possible to change the expression pattern by manipulating the metabolite environment. As a first step in testing this hypothesis, we probed a subset of the intracellular metabolites by partially labeling it with 13C-glucose. We tested 71 genes and identified 17 metabolites by employing the two-dimensional NMR spectroscopy. The statistical analysis showed that there existed the metabolite compositions favoring protein production. We hope that this work would help devise a systematic and predictive approach to the recombinant protein production.

  11. A recombinant fusion protein-based, fluorescent protease assay for high throughput-compatible substrate screening.

    Science.gov (United States)

    Bozóki, Beáta; Gazda, Lívia; Tóth, Ferenc; Miczi, Márió; Mótyán, János András; Tőzsér, József

    2018-01-01

    In connection with the intensive investigation of proteases, several methods have been developed for analysis of the substrate specificity. Due to the great number of proteases and the expected target molecules to be analyzed, time- and cost-efficient high-throughput screening (HTS) methods are preferred. Here we describe the development and application of a separation-based HTS-compatible fluorescent protease assay, which is based on the use of recombinant fusion proteins as substrates of proteases. The protein substrates used in this assay consists of N-terminal (hexahistidine and maltose binding protein) fusion tags, cleavage sequences of the tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein (mApple or mTurquoise2). The assay is based on the fluorimetric detection of the fluorescent proteins, which are released from the magnetic bead-attached substrates by the proteolytic cleavage. The protease assay has been applied for activity measurements of TEV and HIV-1 proteases to test the suitability of the system for enzyme kinetic measurements, inhibition studies, and determination of pH optimum. We also found that denatured fluorescent proteins can be renatured after SDS-PAGE of denaturing conditions, but showed differences in their renaturation abilities. After in-gel renaturation both substrates and cleavage products can be identified by in-gel UV detection. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Case study on human α1-antitrypsin: Recombinant protein titers obtained by commercial ELISA kits are inaccurate

    DEFF Research Database (Denmark)

    Hansen, Henning Gram; Kildegaard, Helene Faustrup; Min Lee, Gyun

    2016-01-01

    Accurate titer determination of recombinant proteins is crucial for evaluating protein production cell lines and processes. Even though enzyme-linked immunosorbent assay (ELISA) is the most widely used assay for determining protein titer, little is known about the accuracy of commercially available...... ELISA kits. We observed that estimations of recombinant human ø1-antitrypsin (rø1AT) titer by Coomassie-stained SDS-PAGE gels did not correspond to previously obtained titers obtained by a commercially available ELISA kit. This prompted us to develop two independent quantification assays based...... on biolayer interferometry and reversed-phase high-performance liquid chromatography. We compared the rø1AT titer obtained by these assays with three different off-the-shelf ELISA kits and found that the ELISA kits led to inconsistent results. The data presented here show that recombinant protein titers...

  13. RYBP Is a K63-Ubiquitin-Chain-Binding Protein that Inhibits Homologous Recombination Repair

    Directory of Open Access Journals (Sweden)

    Mohammad A.M. Ali

    2018-01-01

    Full Text Available Summary: Ring1-YY1-binding protein (RYBP is a member of the non-canonical polycomb repressive complex 1 (PRC1, and like other PRC1 members, it is best described as a transcriptional regulator. However, several PRC1 members were recently shown to function in DNA repair. Here, we report that RYBP preferentially binds K63-ubiquitin chains via its Npl4 zinc finger (NZF domain. Since K63-linked ubiquitin chains are assembled at DNA double-strand breaks (DSBs, we examined the contribution of RYBP to DSB repair. Surprisingly, we find that RYBP is K48 polyubiquitylated by RNF8 and rapidly removed from chromatin upon DNA damage by the VCP/p97 segregase. High expression of RYBP competitively inhibits recruitment of BRCA1 repair complex to DSBs, reducing DNA end resection and homologous recombination (HR repair. Moreover, breast cancer cell lines expressing high endogenous RYBP levels show increased sensitivity to DNA-damaging agents and poly ADP-ribose polymerase (PARP inhibition. These data suggest that RYBP negatively regulates HR repair by competing for K63-ubiquitin chain binding. : Ali et al. find that RYBP binds K63-linked ubiquitin chains and is removed from DNA damage sites. This K63-ubiquitin binding allows RYBP to hinder the recruitment of BRCA1 and Rad51 to DNA double-strand breaks, thus inhibiting homologous recombination repair. Accordingly, cancer cells expressing high RYBP are more sensitive to DNA-damaging therapies. Keywords: DNA damage response, homologous recombination, ubiquitylation, RYBP, polycomb proteins, double-strand break repair, chromatin, histone modification

  14. Application of electrospray mass spectrometry to the characterization of recombinant proteins up to 44 kDa

    NARCIS (Netherlands)

    Van Dorsselaer, A.; Bitsch, F.; Green, B.; Jarvis, S.; Lepage, P.; Bischoff, Rainer; Kolbe, V.J.; Roitsch, C.

    1990-01-01

    Mass measurement by electrospray mass spectrometry (ESMS) is used as a rapid preliminary verification of the identity of various recombinant proteins ranging from 7 to 44 kDa with an accuracy of 0.01-0.03%. ESMS not only improves the speed but also the reliability of the protein structure

  15. Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine.

    Science.gov (United States)

    Kikot, Pamela; Polat, Aise; Achilli, Estefania; Fernandez Lahore, Marcelo; Grasselli, Mariano

    2014-11-01

    Fusion of peptide-based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram-range amounts of proteins. IMAC-Ni(II) columns have become the natural partners of 6xHis-tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His-tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur-containing molecules. In this work, we evaluated two different cysteine- and histidine-containing six amino acid tags linked to the N-terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine-containing tagged GFPs were able to bind to IMAC-Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC-Ni(II) system reaches less than 20% recovery of the cysteine-containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC-Pd(II) yields a recovery of 45% with a purification factor of 13. Copyright © 2014 John Wiley & Sons, Ltd.

  16. "Recombinant Protein of the Day": Using Daily Student Presentations to Add Real-World Aspects to a Biotechnology Course

    Science.gov (United States)

    Shaffer, Justin F.

    2013-01-01

    To provide a realistic view of the biotechnology industry for students, a novel course focusing on recombinant proteins and their importance in medicine, pharmaceuticals, industry, scientific research, and agriculture was developed. ''Designer Proteins and Society,'' an upper-division elective, was taught in the Fall 2012 semester to 16 junior,…

  17. Cloning, expression, and purification of recombinant protein MPT-64 from a virulent strain of Mycobacterium bovis in a prokaryotic system

    Directory of Open Access Journals (Sweden)

    Maryam Mohammadi Tashakkori

    2016-01-01

    Conclusion: These results indicated successful expression and purification of recombinant MPT-64 protein in a prokaryotic system. This protein can be used for serological diagnosis, improved detection of pathogenicity and non-pathogenicity between infected cattle, and for verification of suspected cases of bovine TB.

  18. Modulating secretory pathway pH by proton channel co-expression can increase recombinant protein stability in plants.

    Science.gov (United States)

    Jutras, Philippe V; D'Aoust, Marc-André; Couture, Manon M-J; Vézina, Louis-Philippe; Goulet, Marie-Claire; Michaud, Dominique; Sainsbury, Frank

    2015-09-01

    Eukaryotic expression systems are used for the production of complex secreted proteins. However, recombinant proteins face considerable biochemical challenges along the secretory pathway, including proteolysis and pH variation between organelles. As the use of synthetic biology matures into solutions for protein production, various host-cell engineering approaches are being developed to ameliorate host-cell factors that can limit recombinant protein quality and yield. We report the potential of the influenza M2 ion channel as a novel tool to neutralize the pH in acidic subcellular compartments. Using transient expression in the plant host, Nicotiana benthamiana, we show that ion channel expression can significantly raise pH in the Golgi apparatus and that this can have a strong stabilizing effect on a fusion protein separated by an acid-susceptible linker peptide. We exemplify the utility of this effect in recombinant protein production using influenza hemagglutinin subtypes differentially stable at low pH; the expression of hemagglutinins prone to conformational change in mildly acidic conditions is considerably enhanced by M2 co-expression. The co-expression of a heterologous ion channel to stabilize acid-labile proteins and peptides represents a novel approach to increasing the yield and quality of secreted recombinant proteins in plants and, possibly, in other eukaryotic expression hosts. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Structural and functional characterization of the recombinant death domain from death-associated protein kinase.

    Science.gov (United States)

    Dioletis, Evangelos; Dingley, Andrew J; Driscoll, Paul C

    2013-01-01

    Death-associated protein kinase (DAPk) is a calcium/calmodulin-regulated Ser/Thr-protein kinase that functions at an important point of integration for cell death signaling pathways. DAPk has a structurally unique multi-domain architecture, including a C-terminally positioned death domain (DD) that is a positive regulator of DAPk activity. In this study, recombinant DAPk-DD was observed to aggregate readily and could not be prepared in sufficient yield for structural analysis. However, DAPk-DD could be obtained as a soluble protein in the form of a translational fusion protein with the B1 domain of streptococcal protein G. In contrast to other DDs that adopt the canonical six amphipathic α-helices arranged in a compact fold, the DAPk-DD was found to possess surprisingly low regular secondary structure content and an absence of a stable globular fold, as determined by circular dichroism (CD), NMR spectroscopy and a temperature-dependent fluorescence assay. Furthermore, we measured the in vitro interaction between extracellular-regulated kinase-2 (ERK2) and various recombinant DAPk-DD constructs. Despite the low level of structural order, the recombinant DAPk-DD retained the ability to interact with ERK2 in a 1∶1 ratio with a K d in the low micromolar range. Only the full-length DAPk-DD could bind ERK2, indicating that the apparent 'D-motif' located in the putative sixth helix of DAPk-DD is not sufficient for ERK2 recognition. CD analysis revealed that binding of DAPk-DD to ERK2 is not accompanied by a significant change in secondary structure. Taken together our data argue that the DAPk-DD, when expressed in isolation, does not adopt a classical DD fold, yet in this state retains the capacity to interact with at least one of its binding partners. The lack of a stable globular structure for the DAPk-DD may reflect either that its folding would be supported by interactions absent in our experimental set-up, or a limitation in the structural bioinformatics

  20. Immunogenicity of Recombinant Proteins Consisting of Plasmodium vivax Circumsporozoite Protein Allelic Variant-Derived Epitopes Fused with Salmonella enterica Serovar Typhimurium Flagellin

    Science.gov (United States)

    Leal, Monica Teixeira Andrade; Camacho, Ariane Guglielmi Ariza; Teixeira, Laís Helena; Bargieri, Daniel Youssef; Soares, Irene Silva; Tararam, Cibele Aparecida

    2013-01-01

    A Plasmodium falciparum circumsporozoite protein (CSP)-based recombinant fusion vaccine is the first malaria vaccine to reach phase III clinical trials. Resistance to infection correlated with the production of antibodies to the immunodominant central repeat region of the CSP. In contrast to P. falciparum, vaccine development against the CSP of Plasmodium vivax malaria is far behind. Based on this gap in our knowledge, we generated a recombinant chimeric protein containing the immunodominant central repeat regions of the P. vivax CSP fused to Salmonella enterica serovar Typhimurium-derived flagellin (FliC) to activate the innate immune system. The recombinant proteins that were generated contained repeat regions derived from each of the 3 different allelic variants of the P. vivax CSP or a fusion of regions derived from each of the 3 allelic forms. Mice were subcutaneously immunized with the fusion proteins alone or in combination with the Toll-like receptor 3 (TLR-3) agonist poly(I·C), and the anti-CSP serum IgG response was measured. Immunization with a mixture of the 3 recombinant proteins, each containing immunodominant epitopes derived from a single allelic variant, rather than a single recombinant protein carrying a fusion of regions derived from each of 3 allelic forms elicited a stronger immune response. This response was independent of TLR-4 but required TLR-5/MyD88 activation. Antibody titers significantly increased when poly(I·C) was used as an adjuvant with a mixture of the 3 recombinant proteins. These recombinant fusion proteins are novel candidates for the development of an effective malaria vaccine against P. vivax. PMID:23863502

  1. An All-Recombinant Protein-Based Culture System Specifically Identifies Hematopoietic Stem Cell Maintenance Factors

    Directory of Open Access Journals (Sweden)

    Aki Ieyasu

    2017-03-01

    Full Text Available Hematopoietic stem cells (HSCs are considered one of the most promising therapeutic targets for the treatment of various blood disorders. However, due to difficulties in establishing stable maintenance and expansion of HSCs in vitro, their insufficient supply is a major constraint to transplantation studies. To solve these problems we have developed a fully defined, all-recombinant protein-based culture system. Through this system, we have identified hemopexin (HPX and interleukin-1α as responsible for HSC maintenance in vitro. Subsequent molecular analysis revealed that HPX reduces intracellular reactive oxygen species levels within cultured HSCs. Furthermore, bone marrow immunostaining and 3D immunohistochemistry revealed that HPX is expressed in non-myelinating Schwann cells, known HSC niche constituents. These results highlight the utility of this fully defined all-recombinant protein-based culture system for reproducible in vitro HSC culture and its potential to contribute to the identification of factors responsible for in vitro maintenance, expansion, and differentiation of stem cell populations.

  2. De novo generation of infectious prions with bacterially expressed recombinant prion protein.

    Science.gov (United States)

    Zhang, Zhihong; Zhang, Yi; Wang, Fei; Wang, Xinhe; Xu, Yuanyuan; Yang, Huaiyi; Yu, Guohua; Yuan, Chonggang; Ma, Jiyan

    2013-12-01

    The prion hypothesis is strongly supported by the fact that prion infectivity and the pathogenic conformer of prion protein (PrP) are simultaneously propagated in vitro by the serial protein misfolding cyclic amplification (sPMCA). However, due to sPMCA's enormous amplification power, whether an infectious prion can be formed de novo with bacterially expressed recombinant PrP (rPrP) remains to be satisfactorily resolved. To address this question, we performed unseeded sPMCA with rPrP in a laboratory that has never been exposed to any native prions. Two types of proteinase K (PK)-resistant and self-perpetuating recombinant PrP conformers (rPrP-res) with PK-resistant cores of 17 or 14 kDa were generated. A bioassay revealed that rPrP-res(17kDa) was highly infectious, causing prion disease in wild-type mice with an average survival time of about 172 d. In contrast, rPrP-res(14kDa) completely failed to induce any disease. Our findings reveal that sPMCA is sufficient to initiate various self-perpetuating PK-resistant rPrP conformers, but not all of them possess in vivo infectivity. Moreover, generating an infectious prion in a prion-free environment establishes that an infectious prion can be formed de novo with bacterially expressed rPrP.

  3. Application of recombinant hemagglutinin proteins as alternative antigen standards for pandemic influenza vaccines.

    Science.gov (United States)

    Choi, Yejin; Kwon, Seong Yi; Oh, Ho Jung; Shim, Sunbo; Chang, Seokkee; Chung, Hye Joo; Kim, Do Keun; Park, Younsang; Lee, Younghee

    2017-09-01

    The single radial immunodiffusion (SRID) assay, used to quantify hemagglutinin (HA) in influenza vaccines, requires reference reagents; however, because centralized production of reference reagents may slow the emergency deployment of vaccines, alternatives are needed. We investigated the production of HA proteins using recombinant DNA technology, rather than a traditional egg-based production process. The HA proteins were then used in an SRID assay as a reference antigen. We found that HA can be quantified in both egg-based and cell-based influenza vaccines when recombinant HAs (rHAs) are used as the reference antigen. Furthermore, we confirmed that rHAs obtained from strains with pandemic potential, such as H5N1, H7N3, H7N9, and H9N2 strains, can be utilized in the SRID assay. The rHA production process takes just one month, in contrast to the traditional process that takes three to four months. The use of rHAs may reduce the time required to produce reference reagents and facilitate timely introduction of vaccines during emergencies.

  4. Recombinant proteins of helminths with immunoregulatory properties and their possible therapeutic use.

    Science.gov (United States)

    Nascimento Santos, Leonardo; Carvalho Pacheco, Luis Gustavo; Silva Pinheiro, Carina; Alcantara-Neves, Neuza Maria

    2017-02-01

    The inverse relationship between helminth infections and the development of immune-mediated diseases is a cornerstone of the hygiene hypothesis and studies were carried out to elucidate the mechanisms by which helminth-derived molecules can suppress immunological disorders. These studies have fostered the idea that parasitic worms may be used as a promising therapeutic alternative for prevention and treatment of immune-mediated diseases. We discuss the current approaches for identification of helminth proteins with potential immunoregulatory properties, including the strategies based on high-throughput technologies. We also explore the methodological approaches and expression systems used for production of the recombinant forms of more than 20 helminth immunomodulatory proteins, besides their performances when evaluated as immunotherapeutic molecules to treat different immune-mediated conditions, including asthma and inflammatory bowel diseases. Finally, we discuss the perspectives of using these parasite-derived recombinant molecules as tools for future immunotherapy and immunoprophylaxis of human inflammatory diseases. Copyright © 2016. Published by Elsevier B.V.

  5. Determination of immune status in dogs against CPV-2 by recombinant protein based latex agglutination test.

    Science.gov (United States)

    Thomas, Jobin; Singh, Mithilesh; Goswami, T K; Glora, Philma; Chakravarti, Soumendu; Chander, Vishal; Upmanyu, Vikramaditya; Verma, Suman; Sharma, Chhavi; Mahendran, K

    2017-09-01

    Canine parvoviral enteritis is a highly contagious viral illness caused by canine parvovirus-2 (CPV-2) which affects puppies of mainly 6-20 weeks of age. Vaccination is pivotal in preventing and controlling CPV-2 infection. Determination of antibody status is a critical determinant for successful vaccination. The hemagglutination inhibition (HI) test is 'gold standard' test for quantification of antibodies specific to CPV-2, although the execution of this test is not feasible under field conditions. The present study was undertaken to develop a point of care testing to determine immune status prior to CPV-2 vaccination or to detect seroconversion in immunized dogs by latex agglutination test (LAT) using recombinant antigen. Truncated portion of VP2 protein (tVP2) of CPV-2 was selected on the basis of antigenic indices, overexpressed the recombinant protein in E. coli system and was subsequently used in development of LAT. A total of 59 serum samples obtained from vaccinated (n = 54) and healthy unvaccinated (n = 5) dogs were tested. The positivity was observed in 85% (46/54) of these dogs with varying agglutination pattern. The overall sensitivity and specificity of latex agglutination test in comparison to HI test was recorded as 90% and 88% respectively with an agreement value of 90% (CI = 95%). Copyright © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  6. Production, purification and immunogenicity of recombinant Ebola virus proteins - A comparison of Freund's adjuvant and adjuvant system 03.

    Science.gov (United States)

    Melén, Krister; Kakkola, Laura; He, Felix; Airenne, Kari; Vapalahti, Olli; Karlberg, Helen; Mirazimi, Ali; Julkunen, Ilkka

    2017-04-01

    There is an urgent need for Ebola virus (EBOV) proteins, EBOV-specific antibodies and recombinant antigens to be used in diagnostics and as potential vaccine candidates. Our objective was to produce and purify recombinant proteins for immunological assays and for the production of polyclonal EBOV specific antibodies. In addition, a limited comparison of the adjuvant effects of Freund's complete adjuvant (FCA) and adjuvant system 03 (AS03) was carried out. Recombinant EBOV GST-VP24, -VP30, -VP35, -VP40 and -NP were produced in E. coli and purified with affinity chromatography followed by preparative gel electrophoresis. Recombinant EBOV GP-His was produced in Sf9 insect cells and purified by preparative gel electrophoresis. To compare the adjuvant effect of FCA and AS03, 12 rabbits were immunized four times with one of the six recombinant EBOV proteins using FCA or AS03. In addition, three guinea pigs were immunized with EBOV VP24 using FCA. With the exception of sera from two rabbits immunized with GST-VP24, the antisera against all other EBOV proteins showed very high and specific antibody responses after three to four immunizations. The adjuvant effect of AS03 was comparable to that of FCA. The produced antibodies recognized the corresponding EBOV proteins in wild type EBOV-infected cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. A simple Pichia pastoris fermentation and downstream processing strategy for making recombinant pandemic Swine Origin Influenza a virus Hemagglutinin protein.

    Science.gov (United States)

    Athmaram, T N; Singh, Anil Kumar; Saraswat, Shweta; Srivastava, Saurabh; Misra, Princi; Kameswara Rao, M; Gopalan, N; Rao, P V L

    2013-02-01

    The present Influenza vaccine manufacturing process has posed a clear impediment to initiation of rapid mass vaccination against spreading pandemic influenza. New vaccine strategies are therefore needed that can accelerate the vaccine production. Pichia offers several advantages for rapid and economical bulk production of recombinant proteins and, hence, can be attractive alternative for producing an effective influenza HA based subunit vaccine. The recombinant Pichia harboring the transgene was subjected to fed-batch fermentation at 10 L scale. A simple fermentation and downstream processing strategy is developed for high-yield secretory expression of the recombinant Hemagglutinin protein of pandemic Swine Origin Influenza A virus using Pichia pastoris via fed-batch fermentation. Expression and purification were optimized and the expressed recombinant Hemagglutinin protein was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blot and MALDI-TOF analysis. In this paper, we describe a fed-batch fermentation protocol for the secreted production of Swine Influenza A Hemagglutinin protein in the P. pastoris GS115 strain. We have shown that there is a clear relationship between product yield and specific growth rate. The fed-batch fermentation and downstream processing methods optimized in the present study have immense practical application for high-level production of the recombinant H1N1 HA protein in a cost effective way using P. pastoris.

  8. Polyclonal antibodies against the recombinantly expressed coat protein of the Citrus psorosis virus

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    Reda Salem

    2018-05-01

    Full Text Available Psorosis is a damaging disease of citrus that is widespread in many parts of the world. Citrus psorosis virus (CPsV, the type species of the genus Ophiovirus, is the putative causal agent of psorosis. Detection of CPsV by laboratory methods, serology in particular is a primary requirement for large-scale surveys but their production has been impaired by the difficulty of obtaining sufficient clean antigen for immunization. Specific PAbs against coat protein were produced in E. coli using recombinant DNA approach. The full length CP gene fragment was amplified by RT-PCR using total RNA extracted from CPsV infected citrus leaves and CP specific primers. The obtained product (1320bp was cloned, sequenced and sub-cloned into pET-30(+ expression vector. Expression was induced and screened in different bacterial clones by the presence of the expressed protein (48kDa and optimized in one clone. Expressed CP was purified using batch chromatography under denaturing conditions. Specificity of expressed protein was demonstrated by ELISA before used as antigen for raising PAbs in mice. Specificity of the raised PAbs to CPsV was verified by ELISA and western blotting. The raised PAbs were showed highly effectiveness in screening by ELISA comparing with the commercial antibodies purchased from Agritest, Valanzano, Italy.The expression of CPsV CP gene in E. coli, production of PAbs using recombinant protein as an antigen, the suitability of these antibodies for use in immunodiagnostics against the CPsV Egyptian isolate have been accomplished in this work. Keywords: CPsV, CP, PAbs, RT-PCR, ELISA, Western blotting

  9. Attenuation of Recombinant Yellow Fever 17D Viruses Expressing Foreign Protein Epitopes at the Surface

    Science.gov (United States)

    Bonaldo, Myrna C.; Garratt, Richard C.; Marchevsky, Renato S.; Coutinho, Evandro S. F.; Jabor, Alfredo V.; Almeida, Luís F. C.; Yamamura, Anna M. Y.; Duarte, Adriana S.; Oliveira, Prisciliana J.; Lizeu, Jackeline O. P.; Camacho, Luiz A. B.; Freire, Marcos S.; Galler, Ricardo

    2005-01-01

    The yellow fever (YF) 17D vaccine is a live attenuated virus. Three-dimensional (3D) homology modeling of the E protein structure from YF 17D virus and its comparison with that from tick-borne encephalitis virus revealed that it is possible to accommodate inserts of different sizes and amino acid compositions in the flavivirus E protein fg loop. This is consistent with the 3D structures of both the dimeric and trimeric forms in which the fg loop lies exposed to solvents. We demonstrate here that YF 17D viruses bearing foreign humoral (17D/8) and T-cell (17D/13) epitopes, which vary in sequence and length, displayed growth restriction. It is hypothesized that interference with the dimer-trimer transition and with the formation of a ring of such trimers in order to allow fusion compromises the capability of the E protein to induce fusion of viral and endosomal membranes, and a slower rate of fusion may delay the extent of virus production. This would account for the lower levels of replication in cultured cells and of viremia in monkeys, as well as for the more attenuated phenotype of the recombinant viruses in monkeys. Testing of both recombinant viruses (17D/8 and 17D/13) for monkey neurovirulence also suggests that insertion at the 17D E protein fg loop does not compromise the attenuated phenotype of YF 17D virus, further confirming the potential use of this site for the development of new live attenuated 17D virus-based vaccines. PMID:15956601

  10. Vaccination with Recombinant Microneme Proteins Confers Protection against Experimental Toxoplasmosis in Mice.

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    Camila Figueiredo Pinzan

    Full Text Available Toxoplasmosis, a zoonotic disease caused by Toxoplasma gondii, is an important public health problem and veterinary concern. Although there is no vaccine for human toxoplasmosis, many attempts have been made to develop one. Promising vaccine candidates utilize proteins, or their genes, from microneme organelle of T. gondii that are involved in the initial stages of host cell invasion by the parasite. In the present study, we used different recombinant microneme proteins (TgMIC1, TgMIC4, or TgMIC6 or combinations of these proteins (TgMIC1-4 and TgMIC1-4-6 to evaluate the immune response and protection against experimental toxoplasmosis in C57BL/6 mice. Vaccination with recombinant TgMIC1, TgMIC4, or TgMIC6 alone conferred partial protection, as demonstrated by reduced brain cyst burden and mortality rates after challenge. Immunization with TgMIC1-4 or TgMIC1-4-6 vaccines provided the most effective protection, since 70% and 80% of mice, respectively, survived to the acute phase of infection. In addition, these vaccinated mice, in comparison to non-vaccinated ones, showed reduced parasite burden by 59% and 68%, respectively. The protective effect was related to the cellular and humoral immune responses induced by vaccination and included the release of Th1 cytokines IFN-γ and IL-12, antigen-stimulated spleen cell proliferation, and production of antigen-specific serum antibodies. Our results demonstrate that microneme proteins are potential vaccines against T. gondii, since their inoculation prevents or decreases the deleterious effects of the infection.

  11. Vaccination with Recombinant Microneme Proteins Confers Protection against Experimental Toxoplasmosis in Mice.

    Science.gov (United States)

    Pinzan, Camila Figueiredo; Sardinha-Silva, Aline; Almeida, Fausto; Lai, Livia; Lopes, Carla Duque; Lourenço, Elaine Vicente; Panunto-Castelo, Ademilson; Matthews, Stephen; Roque-Barreira, Maria Cristina

    2015-01-01

    Toxoplasmosis, a zoonotic disease caused by Toxoplasma gondii, is an important public health problem and veterinary concern. Although there is no vaccine for human toxoplasmosis, many attempts have been made to develop one. Promising vaccine candidates utilize proteins, or their genes, from microneme organelle of T. gondii that are involved in the initial stages of host cell invasion by the parasite. In the present study, we used different recombinant microneme proteins (TgMIC1, TgMIC4, or TgMIC6) or combinations of these proteins (TgMIC1-4 and TgMIC1-4-6) to evaluate the immune response and protection against experimental toxoplasmosis in C57BL/6 mice. Vaccination with recombinant TgMIC1, TgMIC4, or TgMIC6 alone conferred partial protection, as demonstrated by reduced brain cyst burden and mortality rates after challenge. Immunization with TgMIC1-4 or TgMIC1-4-6 vaccines provided the most effective protection, since 70% and 80% of mice, respectively, survived to the acute phase of infection. In addition, these vaccinated mice, in comparison to non-vaccinated ones, showed reduced parasite burden by 59% and 68%, respectively. The protective effect was related to the cellular and humoral immune responses induced by vaccination and included the release of Th1 cytokines IFN-γ and IL-12, antigen-stimulated spleen cell proliferation, and production of antigen-specific serum antibodies. Our results demonstrate that microneme proteins are potential vaccines against T. gondii, since their inoculation prevents or decreases the deleterious effects of the infection.

  12. Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli

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    L. Avilán

    1997-12-01

    Full Text Available We cloned the streptokinase (STK gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfate, when a linear gradient was applied. Three major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a Km = 0.70 µM and kcat = 0.82 s-1, when assayed with a chromogen-coupled substrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing

  13. Complex relationship between mismatch repair proteins and MBD4 during immunoglobulin class switch recombination.

    Science.gov (United States)

    Grigera, Fernando; Bellacosa, Alfonso; Kenter, Amy L

    2013-01-01

    Mismatch repair (MMR) safeguards against genomic instability and is required for efficient Ig class switch recombination (CSR). Methyl CpG binding domain protein 4 (MBD4) binds to MutL homologue 1 (MLH1) and controls the post-transcriptional level of several MMR proteins, including MutS homologue 2 (MSH2). We show that in WT B cells activated for CSR, MBD4 is induced and interacts with MMR proteins, thereby implying a role for MBD4 in CSR. However, CSR is in the normal range in Mbd4 deficient mice deleted for exons 2-5 despite concomitant reduction of MSH2. We show by comparison in Msh2(+/-) B cells that a two-fold reduction of MSH2 and MBD4 proteins is correlated with impaired CSR. It is therefore surprising that CSR occurs at normal frequencies in the Mbd4 deficient B cells where MSH2 is reduced. We find that a variant Mbd4 transcript spanning exons 1,6-8 is expressed in Mbd4 deficient B cells. This transcript can be ectopically expressed and produces a truncated MBD4 peptide. Thus, the 3' end of the Mbd4 locus is not silent in Mbd4 deficient B cells and may contribute to CSR. Our findings highlight a complex relationship between MBD4 and MMR proteins in B cells and a potential reconsideration of their role in CSR.

  14. Production and purification of avian antibodies (IgYs from inclusion bodies of a recombinant protein central in NAD+ metabolism

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    Paula A. Moreno-González

    2013-08-01

    Full Text Available The use of hens for the production of polyclonal antibodies reduces animal intervention and moreover yields a higher quantity of antibodies than other animal models.  The phylogenetic distance between bird and mammal antigens, often leads to more specific avian antibodies than their mammalian counterparts.Since a large amount of antigen is required for avian antibody production, the use of recombinant proteins for this procedure has been growing faster over the last years. Nevertheless, recombinant protein production through heterologous systems frequently prompts the protein to precipitate, forming insoluble aggregates of limited utility (inclusion bodies. A methodology for the production of avian polyclonal antibodies, using recombinant protein from inclusion bodies is presented in this article.In order to produce the antigen, a recombinant Nicotinamide mononucleotide adenylyltransferase from Giardia intestinalis (His-GiNMNAT was expressed in Escherichia coli.  The protein was purified through solubilization from inclusion bodies prior to its renaturalization.  Antibodies were purified from egg yolk of immunized hens by water dilution, followed by ammonium sulfate precipitation and thiophilic affinity chromatography.The purified antibodies were tested against His-GiNMNAT protein in Western blot essays. From one egg yolk, 14.4 mg of highly pure IgY were obtained; this antibody was able to detect 15ng of His-GiNMNAT.  IgY specificity was improved by means of antigen affinity purification, allowing its use for parasite protein recognition.

  15. The new pLAI (lux regulon based auto-inducible expression system for recombinant protein production in Escherichia coli

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    Nocadello Salvatore

    2012-01-01

    Full Text Available Abstract Background After many years of intensive research, it is generally assumed that no universal expression system can exist for high-level production of a given recombinant protein. Among the different expression systems, the inducible systems are the most popular for their tight regulation. However, induction is in many cases less favorable due to the high cost and/or toxicity of inducers, incompatibilities with industrial scale-up or detrimental growth conditions. Expression systems using autoinduction (or self-induction prove to be extremely versatile allowing growth and induction of recombinant proteins without the need to monitor cell density or add inducer. Unfortunately, almost all the actual auto inducible expression systems need endogenous or induced metabolic changes during the growth to trigger induction, both frequently linked to detrimental condition to cell growth. In this context, we use a simple modular approach for a cell density-based genetic regulation in order to assemble an autoinducible recombinant protein expression system in E. coli. Result The newly designed pLAI expression system places the expression of recombinant proteins in Escherichia coli under control of the regulatory genes of the lux regulon of Vibrio fischeri's Quorum Sensing (QS system. The pLAI system allows a tight regulation of the recombinant gene allowing a negligible basal expression and expression only at high cell density. Sequence optimization of regulative genes of QS of V. fischeri for expression in E. coli upgraded the system to high level expression. Moreover, partition of regulative genes between the plasmid and the host genome and introduction of a molecular safety lock permitted tighter control of gene expression. Conclusion Coupling gene expression to cell density using cell-to-cell communication provides a promising approach for recombinant protein production. The system allows the control of expression of the target recombinant gene

  16. ACCUMULATION OF RECOMBINANT FUSION PROTEIN – SECRETORY ANALOG OF Ag85B AND ESAT6 MYCOBACTERIUM TUBERCULOSIS PROTEINS – IN TRANSGENIC Lemna minor L. PLANTS

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    A.A.Peterson

    2015-10-01

    Full Text Available Determination of the presence of the recombinant fusion protein (ESAT6-Ag85B(ΔTMD-6His and its accumulation level in duckweed plants (Lemna minor L. was the aim of the research. ESAT6 and Ag85B are secretory proteins of Mycobacterium tuberculosis and are considered as potential candidates for development of new vaccine against tuberculosis (TB. Transgenic duckweed plants were obtained previously by Agrobacterium rhizogenes-mediated transformation and possessed fusion gene sequence esxA-fbpBΔTMD. Specific polyclonal antibodies were produced in immunized mice to identify levels of accumulation of TB antigens in plants. Recombinant antigen used for mice immunization was obtained in our laboratory by expression in E. coli. Western blot analysis revealed the recombinant tuberculosis antigen ESAT6-Ag85B(ΔTMD-6His in extracts from transgenic L. minor plants. The level of accumulation of the protein corresponds to 0.4-0.5 µg protein per 1 g of fresh weight of plant. Additionally, the accumulation of recombinant protein was investigated in lyophilized transgenic plants after 1.5 year storage. Duckweed plants accumulating a recombinant analogue of M. tuberculosis secretory proteins can be used for development of plant-based edible vaccines.

  17. Refolding in high hydrostatic pressure of recombinant proteins from inclusion bodies in Escherichia Coli

    International Nuclear Information System (INIS)

    Balduino, Keli Nunes

    2009-01-01

    The expression of proteins as inclusion bodies in bacteria is a widely used alternative for production of recombinant protein. However, the aggregation is a problem often encountered during refolding of these proteins. High hydrostatic pressure are able to solubilise the inclusion bodies in the presence of low concentrations of denaturant reagents, encouraging refolding protein with high efficiency and reduce costs. This work aims to refolding of recombinant proteins expressed in Escherichia coli from inclusion bodies using high hydrostatic pressure. Three toxins, all featuring five or more disulfide bonds were studied: NXH8, Natterin 2 and Bothropstoxin 1. Suspensions of inclusion bodies of the three proteins were pressurized to 2000 bars for 16 hours. The buffers were optimized for refolding of the three proteins. The buffer used in the refolding of NXH8 was 50 mM Tris HCl, pH 9.0 with proportion of 1GSH: 4GSSG at a concentration of 6 mM and 2 M GdnHCl. Inclusion bodies were used in O.D. (A600nm) of 0.5. After refolding process, dialysis was performed at pH 7.0. The final yield of obtaining soluble NXH8 was 40% (28,6 mg of soluble NXH8/L of culture medium). The refolding of Bothropstoxin 1 was obtained in refolding buffer of Tris HCl 50 mM, pH 7,5 with proportion of 2 GSH: GSSG 3 and concentration of 3 mM and 1 M GdnHCl. Use with a suspension of O.D. (A600nm) of 0.5. The final yield of recovery of Bothropstoxin 1 refolded was 32% (9,2 mg of refolded Bothropstoxin 1/L of culture medium). The refolding of Natterin 2 was performed in the refolding buffer: 20 mM Tris HCl pH 9.0 at a ratio of 2 GSH: 3GSSG and concentration of 10 mM and 1 M GdnHCl and inclusion bodies O.D. (A600nm) of 6.0. The yield of Natterin 2 refolded was 20% (3,7 mg/L of culture medium). Physico-chemical and biological analysis were performed by SDS-PAGE, western blot, scanning electron microscopy, biological tests in vivo and in vitro and structural. The analysis conducted in NXH8 did not show

  18. Biomimetic mineralization of recombinant collagen type I derived protein to obtain hybrid matrices for bone regeneration.

    Science.gov (United States)

    Ramírez-Rodríguez, Gloria Belén; Delgado-López, José Manuel; Iafisco, Michele; Montesi, Monica; Sandri, Monica; Sprio, Simone; Tampieri, Anna

    2016-11-01

    Understanding the mineralization mechanism of synthetic protein has recently aroused great interest especially in the development of advanced materials for bone regeneration. Herein, we propose the synthesis of composite materials through the mineralization of a recombinant collagen type I derived protein (RCP) enriched with RGD sequences in the presence of magnesium ions (Mg) to closer mimic bone composition. The role of both RCP and Mg ions in controlling the precipitation of the mineral phase is in depth evaluated. TEM and X-ray powder diffraction reveal the crystallization of nanocrystalline apatite (Ap) in all the evaluated conditions. However, Raman spectra point out also the precipitation of amorphous calcium phosphate (ACP). This amorphous phase is more evident when RCP and Mg are at work, indicating the synergistic role of both in stabilizing the amorphous precursor. In addition, hybrid matrices are prepared to tentatively address their effectiveness as scaffolds for bone tissue engineering. SEM and AFM imaging show an homogeneous mineral distribution on the RCP matrix mineralized in presence of Mg, which provides a surface roughness similar to that found in bone. Preliminary in vitro tests with pre-osteoblast cell line show good cell-material interaction on the matrices prepared in the presence of Mg. To the best of our knowledge this work represents the first attempt to mineralize recombinant collagen type I derived protein proving the simultaneous effect of the organic phase (RCP) and Mg on ACP stabilization. This study opens the possibility to engineer, through biomineralization process, advanced hybrid matrices for bone regeneration. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Production of a soluble recombinant prion protein fused to blue fluorescent protein without refolding or detergents in Escherichia coli cells.

    Science.gov (United States)

    Arii, Yasuhiro; Yamaguchi, Hidenori; Fukuoka, Shin-Ichi

    2007-10-01

    The physiological function of prion proteins (PrP) remains unclear. To investigate the physiological relevance of PrP, we constructed a fusion protein of PrP with enhanced blue fluorescent protein (PrP-EBFP) to quantify the interaction of PrP with other molecules. Production of soluble PrP-EBFP was achieved by lowering the expression temperature in Escherichia coli (E. coli) cells to 15 degrees C. Soluble PrP-EBFP was purified on cation exchange and heparin-affinity columns to yield high purity protein. This is the first report of the preparation of soluble recombinant PrP without refolding following solubilization using denaturants or disruption using detergents. To confirm the integrity of PrP-EBFP, anisotropy was estimated under physiological conditions in the presence of heparin, which interacts with PrP. The dissociation constant was determined to be 0.88+/-0.07 microM. PrP-EBFP should be useful in the quantification of PrP interactions with other molecules.

  20. GroEL-GroES assisted folding of multiple recombinant proteins simultaneously over-expressed in Escherichia coli.

    Science.gov (United States)

    Goyal, Megha; Chaudhuri, Tapan K

    2015-07-01

    Folding of aggregation prone recombinant proteins through co-expression of chaperonin GroEL and GroES has been a popular practice in the effort to optimize preparation of functional protein in Escherichia coli. Considering the demand for functional recombinant protein products, it is desirable to apply the chaperone assisted protein folding strategy for enhancing the yield of properly folded protein. Toward the same direction, it is also worth attempting folding of multiple recombinant proteins simultaneously over-expressed in E. coli through the assistance of co-expressed GroEL-ES. The genesis of this thinking was originated from the fact that cellular GroEL and GroES assist in the folding of several endogenous proteins expressed in the bacterial cell. Here we present the experimental findings from our study on co-expressed GroEL-GroES assisted folding of simultaneously over-expressed proteins maltodextrin glucosidase (MalZ) and yeast mitochondrial aconitase (mAco). Both proteins mentioned here are relatively larger and aggregation prone, mostly form inclusion bodies, and undergo GroEL-ES assisted folding in E. coli cells during over-expression. It has been reported that the relative yield of properly folded functional forms of MalZ and mAco with the exogenous GroEL-ES assistance were comparable with the results when these proteins were overexpressed alone. This observation is quite promising and highlights the fact that GroEL and GroES can assist in the folding of multiple substrate proteins simultaneously when over-expressed in E. coli. This method might be a potential tool for enhanced production of multiple functional recombinant proteins simultaneously in E. coli. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. A lower isoelectric point increases signal sequence-mediated secretion of recombinant proteins through a bacterial ABC transporter.

    Science.gov (United States)

    Byun, Hyunjong; Park, Jiyeon; Kim, Sun Chang; Ahn, Jung Hoon

    2017-12-01

    Efficient protein production for industrial and academic purposes often involves engineering microorganisms to produce and secrete target proteins into the culture. Pseudomonas fluorescens has a TliDEF ATP-binding cassette transporter, a type I secretion system, which recognizes C-terminal LARD3 signal sequence of thermostable lipase TliA. Many proteins are secreted by TliDEF in vivo when recombined with LARD3, but there are still others that cannot be secreted by TliDEF even when LARD3 is attached. However, the factors that determine whether or not a recombinant protein can be secreted through TliDEF are still unknown. Here, we recombined LARD3 with several proteins and examined their secretion through TliDEF. We found that the proteins secreted via LARD3 are highly negatively charged with highly-acidic isoelectric points (pI) lower than 5.5. Attaching oligo-aspartate to lower the pI of negatively-charged recombinant proteins improved their secretion, and attaching oligo-arginine to negatively-charged proteins blocked their secretion by LARD3. In addition, negatively supercharged green fluorescent protein (GFP) showed improved secretion, whereas positively supercharged GFP did not secrete. These results disclosed that proteins' acidic pI and net negative charge are major factors that determine their secretion through TliDEF. Homology modeling for TliDEF revealed that TliD dimer forms evolutionarily-conserved positively-charged clusters in its pore and substrate entrance site, which also partially explains the pI dependence of the TliDEF-dependent secretions. In conclusion, lowering the isoelectric point improved LARD3-mediated protein secretion, both widening the range of protein targets for efficient production via secretion and signifying an important aspect of ABC transporter-mediated secretions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Construction and Antiapoptosis Activities of Recombinant Adenoviral Expression Vector Carrying EBV Latent Membrane Protein 2A

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    Xishuang Liu

    2011-01-01

    Full Text Available To evaluate the possible effects of LMP2A (EBV latent membrane protein 2A on human gastric cancer cell line SGC-7901, LMP2A coding gene was subcloned into shuttle plasmid pAdTrackCMV to form transfer plasmid pAdTrackCMV-2A, which was linearized with PmeI and cotransformed into E.coli BJ5183 with adenovirus genomic plasmid of pAdeasy-1. The identified recombinant adenovirus plasmid DNA was digested with PacI and transfected into 293 cells to package recombinant adenovirus particles named vAd-2A. Then the expression and antiapoptosis activities of LMP2A on SGC-7901 infected with vAd-2A were analyzed. The vAd-2A was successfully constructed and identified by PCR, restriction digestion, and sequencing. LMP2A expression in SGC was identified by strong green fluorescence expression with fluorescence microscopic photograph and Southern blotting. The growth of LMP2A expressing SGC cells was apparently improved. Both cyclin E expression and S phase ratio in LMP2A expressing SGC cells were upregulated by cell cycle analysis and confocal microscopic analysis respectively. The replication-deficient recombinant adenovirus vector can express LMP2A antigen in SGC cells and inhibit their apoptosis. The results indicate that LMP2A might play an important role in pathogenesis of EBV-associated gastric cancer (EBVaGC. This study establishes a foundation for further study on EBVaGC and its gene therapy.

  3. Recombinant probiotic expressing Listeria adhesion protein attenuates Listeria monocytogenes virulence in vitro.

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    Ok Kyung Koo

    Full Text Available BACKGROUND: Listeria monocytogenes, an intracellular foodborne pathogen, infects immunocompromised hosts. The primary route of transmission is through contaminated food. In the gastrointestinal tract, it traverses the epithelial barrier through intracellular or paracellular routes. Strategies to prevent L. monocytogenes entry can potentially minimize infection in high-risk populations. Listeria adhesion protein (LAP aids L. monocytogenes in crossing epithelial barriers via the paracellular route. The use of recombinant probiotic bacteria expressing LAP would aid targeted clearance of Listeria from the gut and protect high-risk populations from infection. METHODOLOGY/PRINCIPAL FINDINGS: The objective was to investigate the ability of probiotic bacteria or LAP-expressing recombinant probiotic Lactobacillus paracasei (Lbp(LAP to prevent L. monocytogenes adhesion, invasion, and transwell-based transepithelial translocation in a Caco-2 cell culture model. Several wild type probiotic bacteria showed strong adhesion to Caco-2 cells but none effectively prevented L. monocytogenes infection. Pre-exposure to Lbp(LAP for 1, 4, 15, or 24 h significantly (P<0.05 reduced adhesion, invasion, and transepithelial translocation of L. monocytogenes in Caco-2 cells, whereas pre-exposure to parental Lb. paracasei had no significant effect. Similarly, Lbp(LAP pre-exposure reduced L. monocytogenes translocation by as much as 46% after 24 h. Lbp(LAP also prevented L. monocytogenes-mediated cell damage and compromise of tight junction integrity. Furthermore, Lbp(LAP cells reduced L. monocytogenes-mediated cell cytotoxicity by 99.8% after 1 h and 79% after 24 h. CONCLUSIONS/SIGNIFICANCE: Wild type probiotic bacteria were unable to prevent L. monocytogenes infection in vitro. In contrast, Lbp(LAP blocked adhesion, invasion, and translocation of L. monocytogenes by interacting with host cell receptor Hsp60, thereby protecting cells from infection. These data show promise

  4. Serological Assays Based on Recombinant Viral Proteins for the Diagnosis of Arenavirus Hemorrhagic Fevers

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    Masayuki Saijo

    2012-10-01

    Full Text Available The family Arenaviridae, genus Arenavirus, consists of two phylogenetically independent groups: Old World (OW and New World (NW complexes. The Lassa and Lujo viruses in the OW complex and the Guanarito, Junin, Machupo, Sabia, and Chapare viruses in the NW complex cause viral hemorrhagic fever (VHF in humans, leading to serious public health concerns. These viruses are also considered potential bioterrorism agents. Therefore, it is of great importance to detect these pathogens rapidly and specifically in order to minimize the risk and scale of arenavirus outbreaks. However, these arenaviruses are classified as BSL-4 pathogens, thus making it difficult to develop diagnostic techniques for these virus infections in institutes without BSL-4 facilities. To overcome these difficulties, antibody detection systems in the form of an enzyme-linked immunosorbent assay (ELISA and an indirect immunofluorescence assay were developed using recombinant nucleoproteins (rNPs derived from these viruses. Furthermore, several antigen-detection assays were developed. For example, novel monoclonal antibodies (mAbs to the rNPs of Lassa and Junin viruses were generated. Sandwich antigen-capture (Ag-capture ELISAs using these mAbs as capture antibodies were developed and confirmed to be sensitive and specific for detecting the respective arenavirus NPs. These rNP-based assays were proposed to be useful not only for an etiological diagnosis of VHFs, but also for seroepidemiological studies on VHFs. We recently developed arenavirus neutralization assays using vesicular stomatitis virus (VSV-based pseudotypes bearing arenavirus recombinant glycoproteins. The goal of this article is to review the recent advances in developing laboratory diagnostic assays based on recombinant viral proteins for the diagnosis of VHFs and epidemiological studies on the VHFs caused by arenaviruses.

  5. Suppression of cell division by pKi-67 antisense-RNA and recombinant protein.

    Science.gov (United States)

    Duchrow, M; Schmidt, M H; Zingler, M; Anemüller, S; Bruch, H P; Broll, R

    2001-01-01

    The human antigen defined by the monoclonal antibody Ki-67 (pKi-67) is a human nuclear protein strongly associated with cell proliferation and found in all tissues studied. It is widely used as a marker of proliferating cells, yet its function is unknown. To investigate its function we suppressed pKi-67 expression by antisense RNA and overexpressed a partial structure of pKi-67 in HeLa cells. A BrdU-incorporation assay showed a significant decrease in DNA synthesis after antisense inhibition. Cell cycle analysis indicated a higher proportion of cells in G1 phase and a lower proportion of cells in S phase while the number of G(2)/M phase cells remained constant. Overexpression of a recombinant protein encoding three of the repetitive elements from exon 13 of pKi-67 had a similar effect to that obtained by antisense inhibition. The similarity of the effect of expressing 'Ki-67 repeats' and pKi-67 antisense RNA could be explained by a negative effect on the folding of the endogenous protein in the endoplasmatic reticulum. Furthermore excessive self-association of pKi-67 via the repeat structure could inhibit its nuclear transport, preventing it from getting to its presumptive site of action. We conclude that the Ki-67 protein has an important role in the regulation of the cell cycle, which is mediated in part by its repetitive elements. Copyright 2001 S. Karger AG, Basel

  6. Evidence of chemical exchange in recombinant Major Urinary Protein and quenching thereof upon pheromone binding

    Energy Technology Data Exchange (ETDEWEB)

    Perazzolo, Chiara, E-mail: Chiara.Perazzolo@epfl.ch; Verde, Mariachiara [Ecole Polytechnique Federale de Lausanne, Institut des Sciences et Ingenierie Chimiques (Switzerland); Homans, Steve W. [University of Leeds, Institute of Molecular and Cellular Biology (United Kingdom); Bodenhausen, Geoffrey [Ecole Polytechnique Federale de Lausanne, Institut des Sciences et Ingenierie Chimiques (Switzerland)

    2007-05-15

    The internal dynamics of recombinant Major Urinary Protein (rMUP) have been investigated by monitoring transverse nitrogen-15 relaxation using multiple-echo Carr-Purcell-Meiboom-Gill (CPMG) experiments. While the ligand-free protein (APO-rMUP) features extensive evidence of motions on the milliseconds time scale, the complex with 2-methoxy-3-isobutylpyrazine (HOLO-rMUP) appears to be much less mobile on this time scale. At 308 K, exchange rates k{sub ex} = 500-2000 s{sup -1} were typically observed in APO-rMUP for residues located adjacent to a {beta}-turn comprising residues 83-87. These residues occlude an entry to the binding pocket and have been proposed to be a portal for ligand entry in other members of the lipocalin family, such as the retinol binding protein and the human fatty-acid binding protein. Exchange rates and populations are largely uncorrelated, suggesting local 'breathing' motions rather than a concerted global conformational change.

  7. Assessment of the structure of pegylated-recombinant protein therapeutics by the NMR fingerprint assay.

    Science.gov (United States)

    Hodgson, Derek J; Aubin, Yves

    2017-05-10

    A number of recombinant protein therapeutic products, such as filgrastim (methionyl granulocyte colony stimulating factor [Met-GCSF] used to boost the immune system in chemotherapy treated cancer patients), and interferon alpha-2 (used for the treatment of various viral infections), have been chemically modified with the addition of a polyethylene glycol (PEG) chain. This modification prolongs residency of the drug in the body and reduces metabolic degradation, which allows less frequent administration of the products. Here we show how NMR spectroscopy methods can assess the higher order structure (HOS) of pegylated-filgrastim (Neulasta®), pegylated interferon-α2a (Pegasys®) pegylated interferon-α2b (PEG-Intron®) purchased from the marketplace. The addition of the PEG moiety effectively doubles the molecular weight of the three products. This presents a significant challenge for the application of NMR techniques. Nevertheless, the results showed that high-resolution spectra could be recorded for two of the three products. Comparison of the spectra of the pegylated protein and the non-pegylated protein shows that the chemical modification did not alter the HOS of these proteins. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  8. Biotechnology and genetic engineering in the new drug development. Part I. DNA technology and recombinant proteins.

    Science.gov (United States)

    Stryjewska, Agnieszka; Kiepura, Katarzyna; Librowski, Tadeusz; Lochyński, Stanisław

    2013-01-01

    Pharmaceutical biotechnology has a long tradition and is rooted in the last century, first exemplified by penicillin and streptomycin as low molecular weight biosynthetic compounds. Today, pharmaceutical biotechnology still has its fundamentals in fermentation and bioprocessing, but the paradigmatic change affected by biotechnology and pharmaceutical sciences has led to an updated definition. The biotechnology revolution redrew the research, development, production and even marketing processes of drugs. Powerful new instruments and biotechnology related scientific disciplines (genomics, proteomics) make it possible to examine and exploit the behavior of proteins and molecules. Recombinant DNA (rDNA) technologies (genetic, protein, and metabolic engineering) allow the production of a wide range of peptides, proteins, and biochemicals from naturally nonproducing cells. This technology, now approximately 25 years old, is becoming one of the most important technologies developed in the 20(th) century. Pharmaceutical products and industrial enzymes were the first biotech products on the world market made by means of rDNA. Despite important advances regarding rDNA applications in mammalian cells, yeasts still represent attractive hosts for the production of heterologous proteins. In this review we describe these processes.

  9. Effective delivery of recombinant proteins to rod photoreceptors via lipid nanovesicles

    Energy Technology Data Exchange (ETDEWEB)

    Asteriti, Sabrina [Dept. of Translational Research, University of Pisa, Pisa (Italy); Dal Cortivo, Giuditta [Dept. of Life Sciences and Reproduction, University of Verona, Strada Le Grazie 8, Verona (Italy); Pontelli, Valeria [Dept. of Neurological and Movement Sciences, University of Verona, Strada Le Grazie 8, Verona (Italy); Cangiano, Lorenzo [Dept. of Translational Research, University of Pisa, Pisa (Italy); Buffelli, Mario, E-mail: mario.buffelli@univr.it [Dept. of Neurological and Movement Sciences, University of Verona, Strada Le Grazie 8, Verona (Italy); Center for Biomedical Computing, University of Verona, Strada le Grazie 8, 37134 Verona (Italy); Dell’Orco, Daniele, E-mail: daniele.dellorco@univr.it [Dept. of Life Sciences and Reproduction, University of Verona, Strada Le Grazie 8, Verona (Italy); Center for Biomedical Computing, University of Verona, Strada le Grazie 8, 37134 Verona (Italy)

    2015-06-12

    The potential of liposomes to deliver functional proteins in retinal photoreceptors and modulate their physiological response was investigated by two experimental approaches. First, we treated isolated mouse retinas with liposomes encapsulating either recoverin, an important endogenous protein operating in visual phototransduction, or antibodies against recoverin. We then intravitrally injected in vivo liposomes encapsulating either rhodamin B or recoverin and we investigated the distribution in retina sections by confocal microscopy. The content of liposomes was found to be released in higher amount in the photoreceptor layer than in the other regions of the retina and the functional effects of the release were in line with the current model of phototransduction. Our study sets the basis for quantitative investigations aimed at assessing the potential of intraocular protein delivery via biocompatible nanovesicles, with promising implications for the treatment of retinal diseases affecting the photoreceptor layer. - Highlights: • Recombinant proteins encapsulated in nano-sized liposomes injected intravitreally reach retinal photoreceptors. • The phototransduction cascade in rods is modulated by the liposome content. • Mathematical modeling predicts the alteration of the photoresponses following liposome fusion.

  10. N-Lauroylation during the Expression of Recombinant N-Myristoylated Proteins: Implications and Solutions.

    Science.gov (United States)

    Flamm, Andrea Gabriele; Le Roux, Anabel-Lise; Mateos, Borja; Díaz-Lobo, Mireia; Storch, Barbara; Breuker, Kathrin; Konrat, Robert; Pons, Miquel; Coudevylle, Nicolas

    2016-01-01

    Incorporation of myristic acid onto the N terminus of a protein is a crucial modification that promotes membrane binding and correct localization of important components of signaling pathways. Recombinant expression of N-myristoylated proteins in Escherichia coli can be achieved by co-expressing yeast N-myristoyltransferase and supplementing the growth medium with myristic acid. However, undesired incorporation of the 12-carbon fatty acid lauric acid can also occur (leading to heterogeneous samples), especially when the available carbon sources are scarce, as it is the case in minimal medium for the expression of isotopically enriched samples. By applying this method to the brain acid soluble protein 1 and the 1-185 N-terminal region of c-Src, we show the significant, and protein-specific, differences in the membrane binding properties of lauroylated and myristoylated forms. We also present a robust strategy for obtaining lauryl-free samples of myristoylated proteins in both rich and minimal media. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  11. Evidence of chemical exchange in recombinant Major Urinary Protein and quenching thereof upon pheromone binding

    International Nuclear Information System (INIS)

    Perazzolo, Chiara; Verde, Mariachiara; Homans, Steve W.; Bodenhausen, Geoffrey

    2007-01-01

    The internal dynamics of recombinant Major Urinary Protein (rMUP) have been investigated by monitoring transverse nitrogen-15 relaxation using multiple-echo Carr-Purcell-Meiboom-Gill (CPMG) experiments. While the ligand-free protein (APO-rMUP) features extensive evidence of motions on the milliseconds time scale, the complex with 2-methoxy-3-isobutylpyrazine (HOLO-rMUP) appears to be much less mobile on this time scale. At 308 K, exchange rates k ex = 500-2000 s -1 were typically observed in APO-rMUP for residues located adjacent to a β-turn comprising residues 83-87. These residues occlude an entry to the binding pocket and have been proposed to be a portal for ligand entry in other members of the lipocalin family, such as the retinol binding protein and the human fatty-acid binding protein. Exchange rates and populations are largely uncorrelated, suggesting local 'breathing' motions rather than a concerted global conformational change

  12. Production of Polyclonal Antibodies to the Recombinant Potato virus M (PVM) Non-structural Triple Gene Block Protein 1 and Coat Protein

    Czech Academy of Sciences Publication Activity Database

    Čeřovská, Noemi; Moravec, Tomáš; Plchová, Helena; Hoffmeisterová, Hana; Dědič, P.

    2012-01-01

    Roč. 160, č. 5 (2012), s. 251-254 ISSN 0931-1785 R&D Projects: GA MŠk 1M06030 Institutional research plan: CEZ:AV0Z50380511 Keywords : Potato virus M * recombinant protein * coat protein Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.000, year: 2012

  13. Recombination Rate Heterogeneity within Arabidopsis Disease Resistance Genes.

    Science.gov (United States)

    Choi, Kyuha; Reinhard, Carsten; Serra, Heïdi; Ziolkowski, Piotr A; Underwood, Charles J; Zhao, Xiaohui; Hardcastle, Thomas J; Yelina, Nataliya E; Griffin, Catherine; Jackson, Matthew; Mézard, Christine; McVean, Gil; Copenhaver, Gregory P; Henderson, Ian R

    2016-07-01

    Meiotic crossover frequency varies extensively along chromosomes and is typically concentrated in hotspots. As recombination increases genetic diversity, hotspots are predicted to occur at immunity genes, where variation may be beneficial. A major component of plant immunity is recognition of pathogen Avirulence (Avr) effectors by resistance (R) genes that encode NBS-LRR domain proteins. Therefore, we sought to test whether NBS-LRR genes would overlap with meiotic crossover hotspots using experimental genetics in Arabidopsis thaliana. NBS-LRR genes tend to physically cluster in plant genomes; for example, in Arabidopsis most are located in large clusters on the south arms of chromosomes 1 and 5. We experimentally mapped 1,439 crossovers within these clusters and observed NBS-LRR gene associated hotspots, which were also detected as historical hotspots via analysis of linkage disequilibrium. However, we also observed NBS-LRR gene coldspots, which in some cases correlate with structural heterozygosity. To study recombination at the fine-scale we used high-throughput sequencing to analyze ~1,000 crossovers within the RESISTANCE TO ALBUGO CANDIDA1 (RAC1) R gene hotspot. This revealed elevated intragenic crossovers, overlapping nucleosome-occupied exons that encode the TIR, NBS and LRR domains. The highest RAC1 recombination frequency was promoter-proximal and overlapped CTT-repeat DNA sequence motifs, which have previously been associated with plant crossover hotspots. Additionally, we show a significant influence of natural genetic variation on NBS-LRR cluster recombination rates, using crosses between Arabidopsis ecotypes. In conclusion, we show that a subset of NBS-LRR genes are strong hotspots, whereas others are coldspots. This reveals a complex recombination landscape in Arabidopsis NBS-LRR genes, which we propose results from varying coevolutionary pressures exerted by host-pathogen relationships, and is influenced by structural heterozygosity.

  14. Efficient Secretion of Recombinant Proteins from Rice Suspension-Cultured Cells Modulated by the Choice of Signal Peptide.

    Science.gov (United States)

    Huang, Li-Fen; Tan, Chia-Chun; Yeh, Ju-Fang; Liu, Hsin-Yi; Liu, Yu-Kuo; Ho, Shin-Lon; Lu, Chung-An

    2015-01-01

    Plant-based expression systems have emerged as a competitive platform in the large-scale production of recombinant proteins. By adding a signal peptide, αAmy3sp, the desired recombinant proteins can be secreted outside transgenic rice cells, making them easy to harvest. In this work, to improve the secretion efficiency of recombinant proteins in rice expression systems, various signal peptides including αAmy3sp, CIN1sp, and 33KDsp have been fused to the N-terminus of green fluorescent protein (GFP) and introduced into rice cells to explore the efficiency of secretion of foreign proteins. 33KDsp had better efficiency than αAmy3sp and CIN1sp for the secretion of GFP from calli and suspension-cultured cells. 33KDsp was further applied for the secretion of mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) from transgenic rice suspension-cultured cells; approximately 76%-92% of total rice-derived mGM-CSF (rmGM-CSF) was detected in the culture medium. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60. The extracellular yield of rmGM-CSF reached 31.7 mg/L. Our study indicates that 33KDsp is better at promoting the secretion of recombinant proteins in rice suspension-cultured cell systems than the commonly used αAmy3sp.

  15. Transfection of primary brain capillary endothelial cells for protein synthesis and secretion of recombinant erythropoietin: a strategy to enable protein delivery to the brain

    DEFF Research Database (Denmark)

    Burkhart, Annette; Andresen, Thomas Lars; Aigner, Achim

    2017-01-01

    , as turning BCECs into recombinant protein factories by transfection could result in protein secretion further into the brain. The present study aims to investigate the possibility of transfecting primary rat brain endothelial cells (RBECs) for recombinant protein synthesis and secretion...... of the neuroprotective protein erythropoietin (EPO). We previously showed that 4% of RBECs with BBB properties can be transfected without disrupting the BBB integrity in vitro, but it can be questioned whether this is sufficient to enable protein secretion at therapeutic levels. The present study examined various......-derived neurotropic factor (BDNF). In conclusion, non-viral gene therapy to RBECs leads to protein secretion and signifies a method for therapeutic proteins to target cells inside the CNS otherwise omitted due to the BBB....

  16. Production of functional human insulin-like growth factor binding proteins (IGFBPs) using recombinant expression in HEK293 cells.

    Science.gov (United States)

    Wanscher, Anne Sofie Molsted; Williamson, Michael; Ebersole, Tasja Wainani; Streicher, Werner; Wikström, Mats; Cazzamali, Giuseppe

    2015-04-01

    Insulin-like growth factor binding proteins (IGFBPs) display many functions in humans including regulation of the insulin-like growth factor (IGF) signaling pathway. The various roles of human IGFBPs make them attractive protein candidates in drug discovery. Structural and functional knowledge on human proteins with therapeutic relevance is needed to design and process the next generation of protein therapeutics. In order to conduct structural and functional investigations large quantities of recombinant proteins are needed. However, finding a suitable recombinant production system for proteins such as full-length human IGFBPs, still remains a challenge. Here we present a mammalian HEK293 expression method suitable for over-expression of secretory full-length human IGFBP-1 to -7. Protein purification of full-length human IGFBP-1, -2, -3 and -5 was conducted using a two-step chromatography procedure and the final protein yields were between 1 and 12mg protein per liter culture media. The recombinant IGFBPs contained PTMs and exhibited high-affinity interactions with their natural ligands IGF-1 and IGF-2. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Comparative evaluation of the diagnostic potential of recombinant envelope proteins and native cell culture purified viral antigens of Chikungunya virus.

    Science.gov (United States)

    Khan, Mohsin; Dhanwani, Rekha; Kumar, Jyoti S; Rao, P V Lakshmana; Parida, Manmohan

    2014-07-01

    Despite the fact that Chikungunya resurgence is associated with epidemic of unprecedented magnitude, there are challenges in the field of its clinical diagnosis. However, serological tests in an ELISA format provide a rapid tool for the diagnosis of Chikungunya infection. Indeed, ELISAs based on recombinant proteins hold a great promise as these methods are cost effective and are free from the risk of handling biohazardous material. In this study, the performance of recombinant CHIKV antigens was compared in various ELISA formats for the diagnosis of Chikungunya. Two recombinant antigens derived from the envelope proteins of Chikungunya virus were prepared and evaluated by comparing their competence for detecting circulating antibodies in serum samples of patients infected with CHIKV using MAC-ELISA and indirect IgM-ELISA. The efficacy of the recombinant antigens was also compared with the native antigen. The indirect antibody capture IgM microplate ELISA revealed ≥90% concordance with the native antigen in detecting the CHIKV specific IgM antibodies whereas the recombinant antigen based MAC-ELISA showed 100% specificity. The recombinant antigens used in this study were effective and reliable targets for the diagnosis of CHIKV infection and also provide an alternative for native antigen use which is potentially biohazardous. © 2013 Wiley Periodicals, Inc.

  18. p53 Protein interacts specifically with the meiosis-specific mammalian RecA-like protein DMC1 in meiosis.

    Science.gov (United States)

    Habu, Toshiyuki; Wakabayashi, Nobunao; Yoshida, Kayo; Yomogida, Kenntaro; Nishimune, Yoshitake; Morita, Takashi

    2004-06-01

    The tumor suppressor protein p53 is specifically expressed during meiosis in spermatocytes. Subsets of p53 knockout mice exhibit testicular giant cell degenerative syndrome, which suggests p53 may be associated with meiotic cell cycle and/or DNA metabolism. Here, we show that p53 binds to the mouse meiosis-specific RecA-like protein Mus musculus DMC1 (MmDMC1). The C-terminal domain (amino acid 234-340) of MmDMC1 binds to DNA-binding domain of p53 protein. p53 might be involved in homologous recombination and/or checkpoint function by directly binding to DMC1 protein to repress genomic instability in meiotic germ cells.

  19. Kosmotropic anions promote conversion of recombinant prion protein into a PrPSc-like misfolded form.

    Directory of Open Access Journals (Sweden)

    Rodrigo Diaz-Espinoza

    Full Text Available Prions are self-propagating proteins involved in transmissible spongiform encephalopaties in mammals. An aberrant conformation with amyloid-like features of a cell surface protein, termed prion protein (PrP, is thought to be the essential component of the infectious particle, though accessory co-factor molecules such as lipids and nucleotides may be involved. The cellular co-factors and environmental conditions implicated in PrP misfolding are not completely understood. To address this issue, several studies have been done inducing misfolding of recombinant PrP (recPrP into classical amyloid structures using partially denaturing conditions. In this work, we report that misfolding of recPrP into PrP(Sc-like aggregates can be induced by simply incubating the protein in the presence of kosmotropic salts at concentrations that are known to retain or increase the stability of the protein. We used a simple experimental reaction (protein, buffer and salts submitted to agitation/incubation cycles at physiological temperature and pH. The formation of protease resistant-recPrP was time and salt-concentration dependent and required the presence of kosmotropic anions such as F(- or SO(4(-2. The molecular weights of the protease resistant recPrP fragments are reminiscent of those found in degradation assays of bona fide PrP(Sc. The aggregates also exhibited PrP(Sc-like ultrastructural features including rod-shape morphology under electron microscope, high beta-sheet content and thioflavin-T positive signal. The formation of recPrP aggregates with PrP(Sc biochemical features under conditions closer to physiological in the absence of organic co-factor molecules provides a simple setup that may prove helpful to understand the molecular mechanism of PrP misfolding.

  20. Functional Assembly of Soluble and Membrane Recombinant Proteins of Mammalian NADPH Oxidase Complex.

    Science.gov (United States)

    Souabni, Hajer; Ezzine, Aymen; Bizouarn, Tania; Baciou, Laura

    2017-01-01

    Activation of phagocyte cells from an innate immune system is associated with a massive consumption of molecular oxygen to generate highly reactive oxygen species (ROS) as microbial weapons. This is achieved by a multiprotein complex, the so-called NADPH oxidase. The activity of phagocyte NADPH oxidase relies on an assembly of more than five proteins, among them the membrane heterodimer named flavocytochrome b 558 (Cytb 558 ), constituted by the tight association of the gp91 phox (also named Nox2) and p22 phox proteins. The Cytb 558 is the membrane catalytic core of the NADPH oxidase complex, through which the reducing equivalent provided by NADPH is transferred via the associated prosthetic groups (one flavin and two hemes) to reduce dioxygen into superoxide anion. The other major proteins (p47 phox , p67 phox , p40 phox , Rac) requisite for the complex activity are cytosolic proteins. Thus, the NADPH oxidase functioning relies on a synergic multi-partner assembly that in vivo can be hardly studied at the molecular level due to the cell complexity. Thus, a cell-free assay method has been developed to study the NADPH oxidase activity that allows measuring and eventually quantifying the ROS generation based on optical techniques following reduction of cytochrome c. This setup is a valuable tool for the identification of protein interactions, of crucial components and additives for a functional enzyme. Recently, this method was improved by the engineering and the production of a complete recombinant NADPH oxidase complex using the combination of purified proteins expressed in bacterial and yeast host cells. The reconstitution into artificial membrane leads to a fully controllable system that permits fine functional studies.

  1. Engineer Medium and Feed for Modulating N-Glycosylation of Recombinant Protein Production in CHO Cell Culture.

    Science.gov (United States)

    Fan, Yuzhou; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

    2017-01-01

    Chinese hamster ovary (CHO) cells have become the primary expression system for the production of complex recombinant proteins due to their long-term success in industrial scale production and generating