Spider Silk Fibers Spun from Soluble Recombinant Silk Produced in Mammalian Cells

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Lazaris, Anthoula; Arcidiacono, Steven; Huang, Yue; Zhou, Jiang-Feng; Duguay, François; Chretien, Nathalie; Welsh, Elizabeth A.; Soares, Jason W.; Karatzas, Costas N.

2002-01-01

Spider silks are protein-based ``biopolymer'' filaments or threads secreted by specialized epithelial cells as concentrated soluble precursors of highly repetitive primary sequences. Spider dragline silk is a flexible, lightweight fiber of extraordinary strength and toughness comparable to that of synthetic high-performance fibers. We sought to ``biomimic'' the process of spider silk production by expressing in mammalian cells the dragline silk genes (ADF-3/MaSpII and MaSpI) of two spider species. We produced soluble recombinant (rc)-dragline silk proteins with molecular masses of 60 to 140 kilodaltons. We demonstrated the wet spinning of silk monofilaments spun from a concentrated aqueous solution of soluble rc-spider silk protein (ADF-3; 60 kilodaltons) under modest shear and coagulation conditions. The spun fibers were water insoluble with a fine diameter (10 to 40 micrometers) and exhibited toughness and modulus values comparable to those of native dragline silks but with lower tenacity. Dope solutions with rc-silk protein concentrations >20% and postspinning draw were necessary to achieve improved mechanical properties of the spun fibers. Fiber properties correlated with finer fiber diameter and increased birefringence.

Expression and Purification of Recombinant Proteins in Escherichia coli with a His6 or Dual His6-MBP Tag.

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Raran-Kurussi, Sreejith; Waugh, David S

2017-01-01

Rapid advances in bioengineering and biotechnology over the past three decades have greatly facilitated the production of recombinant proteins in Escherichia coli. Affinity-based methods that employ protein or peptide based tags for protein purification have been instrumental in this progress. Yet insolubility of recombinant proteins in E. coli remains a persistent problem. One way around this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) is widely acknowledged as a highly effective solubilizing agent. In this chapter, we describe how to construct either a His 6 - or a dual His 6 -MBP tagged fusion protein by Gateway ® recombinational cloning and how to evaluate their yield and solubility. We also describe a simple and rapid procedure to test the solubility of proteins after removing their N-terminal fusion tags by tobacco etch virus (TEV) protease digestion. The choice of whether to use a His 6 tag or a His 6 -MBP tag can be made on the basis of this solubility test.

Stabilization of apoglobin by low temperature increases yield of soluble recombinant hemoglobin in Escherichia coli.

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Weickert, M J; Pagratis, M; Curry, S R; Blackmore, R

1997-01-01

Accumulation of soluble recombinant hemoglobin (rHb1.1) in Escherichia coli requires proper protein folding, prosthetic group (heme) addition, and subunit assembly. This served as a new model system for the study of the effects of temperature, protein synthesis rates, and protein accumulation rates on protein solubility in E. coli. Fermentation expression of rHb1.1 at 30 degrees C from cultures containing a medium or high globin gene dosage (pBR-based or pUC-based plasmids with rHb1.1 genes under the control of the tac promoter) was compared. A medium gene dosage resulted in rHb1.1 accumulating to approximately 7% of the soluble cell protein, of which 78% was soluble. A high globin gene dosage resulted in a > or = 3-fold increase in total globin to 23 to 24% of the soluble cell protein, but 70% was insoluble. Accumulation of insoluble rHb1.1 began immediately upon induction. The proportion of rHb1.1 from the high globin gene dosage that accumulated as insoluble globin was affected by reducing (i) the inducer concentration and (ii) the temperature. Reducing the inducer concentration reduced globin synthesis up to eightfold but increased the proportion of soluble rHb1.1 to 93%. In contrast, total globin protein synthesis was barely affected by reducing the temperature from 30 to 26 degrees C, while soluble globin accumulation increased > 2-fold to approximately 15% of the soluble cell protein. The contrast between the effects of reducing rates of protein synthesis and accumulation and those of reducing temperature suggests that lower temperature stabilizes one or more folding intermediates. We propose a simplified physical model which integrates protein synthesis, folding, and heme association. This model shows that temperature-dependent apoglobin stability is the most critical factor in soluble rHb1.1 accumulation. PMID:9361418

High-level expression and purification of soluble recombinant FGF21 protein by SUMO fusion in Escherichia coli

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Huang Yadong

2010-02-01

Full Text Available Abstract Background Fibroblast growth factor 21 (FGF21 is a promising drug candidate to combat metabolic diseases. However, high-level expression and purification of recombinant FGF21 (rFGF21 in Escherichia coli (E. coli is difficult because rFGF21 forms inclusion bodies in the bacteria making it difficult to purify and obtain high concentrations of bioactive rFGF21. To overcome this problem, we fused the FGF21 with SUMO (Small ubiquitin-related modifier by polymerase chain reaction (PCR, and expressed the fused gene in E. coli BL21(DE3. Results By inducing with IPTG, SUMO-FGF21 was expressed at a high level. Its concentration reached 30% of total protein, and exceeded 95% of all soluble proteins. The fused protein was purified by DEAE sepharose FF and Ni-NTA affinity chromatography. Once cleaved by the SUMO protease, the purity of rFGF21 by high performance liquid chromatography (HPLC was shown to be higher than 96% with low endotoxin level (in vivo animal experiments showed that rFGF21 produced by using this method, could decrease the concentration of plasma glucose in diabetic rats by streptozotocin (STZ injection. Conclusions This study demonstrated that SUMO, when fused with FGF21, was able to promote its soluble expression of the latter in E. coli, making it more convenient to purify rFGF21 than previously. This may be a better method to produce rFGF21 for pharmaceutical research and development.

Recombinant protein production data after expression in the bacterium Escherichia coli

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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.

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

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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.

Soluble expression of recomb inant cMyc, Klf4, Oct4, and Sox2 proteins in bacteria and transduction into living cells

Directory of Open Access Journals (Sweden)

Guo-Dan Liu

2017-04-01

Full Text Available AIM: To develop a new method to produce recombinant reprogramming proteins, cMyc, Klf4, Oct4, and Sox2, in soluble format with low cost for the generation of induced pluripotent stem cells (iPSCs. METHODS: A short polypeptide sequence derived from the HIV trans-activator of transcription protein (TAT and the nucleus localization signal (NLS polypeptide were fused to the N terminus of the reprogramming proteins and they were constructed into pCold-SUMO vector which can extremely improve the solubility of recombinant proteins. Then these vector plasmids were transformed into E. coli BL21 (DE3 Chaperone competent cells for amplification. The solubility of these recombinant proteins was determined by SDS-PAGE and Coomassie brilliant blue staining. The recombinant proteins were purified by Ni-NTA resin and identified by Western blot. The transduction of these proteins into HEK 293T cells were evaluated by immunofluorescence staining. RESULTS: These four reprogramming proteins could be produced in soluble format in pCold-SUMO expression vector system with the assistance of chaperone proteins in bacteria. The proteins were purified successfully with a purity of over 70% with a relative high transduction rate into 293 cells. CONCLUSION: The results in the present study indicate the four important reprogramming proteins, cMyc, Klf4, Oct4, and Sox2, can be produced in soluble format in bacteria with low cost. Our new method thus might be expected to greatly contribute to the future study of iPSCs.

Expression and Purification of Soluble STAT5b/STAT3 Proteins for SH2 Domain Binding Assay.

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Asai, Akira; Takakuma, Kazuyuki

2017-01-01

When a large hydrophobic full-length protein is expressed in bacteria, it is often challenging to obtain recombinant proteins in the soluble fraction. One way to overcome this challenge is expression of deletion mutants that have improved solubility while maintaining biological activity. In this chapter, we describe a protocol for expression of truncated forms of STAT5b and STAT3 proteins that are soluble and retain SH2-mediated activity for phospho-Tyr peptide recognition.

PON-Sol: prediction of effects of amino acid substitutions on protein solubility.

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Yang, Yang; Niroula, Abhishek; Shen, Bairong; Vihinen, Mauno

2016-07-01

Solubility is one of the fundamental protein properties. It is of great interest because of its relevance to protein expression. Reduced solubility and protein aggregation are also associated with many diseases. We collected from literature the largest experimentally verified solubility affecting amino acid substitution (AAS) dataset and used it to train a predictor called PON-Sol. The predictor can distinguish both solubility decreasing and increasing variants from those not affecting solubility. PON-Sol has normalized correct prediction ratio of 0.491 on cross-validation and 0.432 for independent test set. The performance of the method was compared both to solubility and aggregation predictors and found to be superior. PON-Sol can be used for the prediction of effects of disease-related substitutions, effects on heterologous recombinant protein expression and enhanced crystallizability. One application is to investigate effects of all possible AASs in a protein to aid protein engineering. PON-Sol is freely available at http://structure.bmc.lu.se/PON-Sol The training and test data are available at http://structure.bmc.lu.se/VariBench/ponsol.php mauno.vihinen@med.lu.se Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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

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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.

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

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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.

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

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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.

Efficient protein production method for NMR using soluble protein tags with cold shock expression vector

International Nuclear Information System (INIS)

Hayashi, Kokoro; Kojima, Chojiro

2010-01-01

The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was investigated. The pCold-GST system successfully expressed 9 out of 10 proteins that otherwise could not be expressed using a conventional E. coli expression system. Here, we applied the pCold-GST system to 84 proteins and 78 proteins were successfully expressed in the soluble fraction. Three other cold-shock expression systems containing a maltose binding protein tag (pCold-MBP), protein G B1 domain tag (pCold-GB1) or thioredoxin tag (pCold-Trx) were also developed to improve the yield. Additionally, we show that a C-terminal proline tag, which is invisible in 1 H- 15 N HSQC spectra, inhibits protein degradation and increases the final yield of unstable proteins. The purified proteins were amenable to NMR analyses. These data suggest that pCold expression systems combined with soluble protein tags can be utilized to improve the expression and purification of various proteins for NMR analysis.

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

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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.

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...

Efficient protein production method for NMR using soluble protein tags with cold shock expression vector

Energy Technology Data Exchange (ETDEWEB)

Hayashi, Kokoro [Fujifilm Corporation, Analysis Technology Center (Japan); Kojima, Chojiro, E-mail: kojima@protein.osaka-u.ac.j [Nara Institute of Science and Technology (NAIST), Graduate School of Biological Sciences (Japan)

2010-11-15

The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was investigated. The pCold-GST system successfully expressed 9 out of 10 proteins that otherwise could not be expressed using a conventional E. coli expression system. Here, we applied the pCold-GST system to 84 proteins and 78 proteins were successfully expressed in the soluble fraction. Three other cold-shock expression systems containing a maltose binding protein tag (pCold-MBP), protein G B1 domain tag (pCold-GB1) or thioredoxin tag (pCold-Trx) were also developed to improve the yield. Additionally, we show that a C-terminal proline tag, which is invisible in {sup 1}H-{sup 15}N HSQC spectra, inhibits protein degradation and increases the final yield of unstable proteins. The purified proteins were amenable to NMR analyses. These data suggest that pCold expression systems combined with soluble protein tags can be utilized to improve the expression and purification of various proteins for NMR analysis.

Efficient expression of a soluble lipid transfer protein (LTP) of Platanus orientalis using short peptide tags and structural comparison with the natural form.

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Salari, Farhad; Vahedi, Fatemeh; Chamani, Jamshidkhan; Varasteh, Abdolreza; Ketabdar, Hanieh; Sankian, Mojtaba

2015-01-01

Successful recombinant allergen-based immunotherapy has drawn a great deal of attention to use recombinant allergens for new therapeutic and/or diagnostic strategies. The Escherichia coli expression system is frequently used to produce recombinant allergens; however, protein expression in E. coli often results in inclusion bodies. Here, we focused on the expression of two recombinant soluble forms of Pla or 3 using solubility-enhancing peptide tags, human immune deficiency virus type 1 transactivator of transcription core domain and poly-arginine-lysine: rTAT-Pla or 3 and rPoly-Arg-Lys-Pla or 3. Structural characteristics and IgE reactivity of purified recombinant proteins were compared with natural Pla or 3 (nPla or 3) isolated from Platanus orientalis using circular dichroism spectra, fluorescence spectroscopy, and immunoblotting. Likewise, intrinsic viscosity and Stokes radius of the natural and recombinant Pla or 3 allergens were determined to analyze structural compactness in aqueous media. The results indicate high-level solubility and efficient expression of the fusion proteins (rTAT-Pla or 3 and rPoly-Arg-Lys-Pla or 3) compared with the wild-type recombinant. Furthermore, the similar structural characteristics and IgE-binding activities of the fusion proteins to nPla or 3 provide a promising tool for allergy diagnosis and treatment. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.

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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.

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...

How to find soluble proteins: a comprehensive analysis of alpha/beta hydrolases for recombinant expression in E. coli

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Barth Sandra

2005-04-01

Full Text Available Abstract Background In screening of libraries derived by expression cloning, expression of active proteins in E. coli can be limited by formation of inclusion bodies. In these cases it would be desirable to enrich gene libraries for coding sequences with soluble gene products in E. coli and thus to improve the efficiency of screening. Previously Wilkinson and Harrison showed that solubility can be predicted from amino acid composition (Biotechnology 1991, 9(5:443–448. We have applied this analysis to members of the alpha/beta hydrolase fold family to predict their solubility in E. coli. alpha/beta hydrolases are a highly diverse family with more than 1800 proteins which have been grouped into homologous families and superfamilies. Results The predicted solubility in E. coli depends on hydrolase size, phylogenetic origin of the host organism, the homologous family and the superfamily, to which the hydrolase belongs. In general small hydrolases are predicted to be more soluble than large hydrolases, and eukaryotic hydrolases are predicted to be less soluble in E. coli than prokaryotic ones. However, combining phylogenetic origin and size leads to more complex conclusions. Hydrolases from prokaryotic, fungal and metazoan origin are predicted to be most soluble if they are of small, medium and large size, respectively. We observed large variations of predicted solubility between hydrolases from different homologous families and from different taxa. Conclusion A comprehensive analysis of all alpha/beta hydrolase sequences allows more efficient screenings for new soluble alpha/beta hydrolases by the use of libraries which contain more soluble gene products. Screening of hydrolases from families whose members are hard to express as soluble proteins in E. coli should first be done in coding sequences of organisms from phylogenetic groups with the highest average of predicted solubility for proteins of this family. The tools developed here can be used

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

A successful strategy for the recovering of active P21, an insoluble recombinant protein of Trypanosoma cruzi

Science.gov (United States)

Santos, Marlus Alves Dos; Teixeira, Francesco Brugnera; Moreira, Heline Hellen Teixeira; Rodrigues, Adele Aud; Machado, Fabrício Castro; Clemente, Tatiana Mordente; Brigido, Paula Cristina; Silva, Rebecca Tavares E.; Purcino, Cecílio; Gomes, Rafael Gonçalves Barbosa; Bahia, Diana; Mortara, Renato Arruda; Munte, Claudia Elisabeth; Horjales, Eduardo; da Silva, Claudio Vieira

2014-03-01

Structural studies of proteins normally require large quantities of pure material that can only be obtained through heterologous expression systems and recombinant technique. In these procedures, large amounts of expressed protein are often found in the insoluble fraction, making protein purification from the soluble fraction inefficient, laborious, and costly. Usually, protein refolding is avoided due to a lack of experimental assays that can validate correct folding and that can compare the conformational population to that of the soluble fraction. Herein, we propose a validation method using simple and rapid 1D 1H nuclear magnetic resonance (NMR) spectra that can efficiently compare protein samples, including individual information of the environment of each proton in the structure.

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

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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.

A new tagging system for production of recombinant proteins in Drosophila S2 cells using the third domain of the urokinase receptor

DEFF Research Database (Denmark)

Gårdsvoll, Henrik; Hansen, Line V; Jørgensen, Thomas J D

2007-01-01

The use of protein fusion tag technology greatly facilitates detection, expression and purification of recombinant proteins, and the demands for new and more effective systems are therefore expanding. We have used a soluble truncated form of the third domain of the urokinase receptor...... as a convenient C-terminal fusion partner for various recombinant extracellular human proteins used in basic cancer research. The stability of this cystein-rich domain, which structure adopts a three-finger fold, provides an important asset for its applicability as a fusion tag for expression of recombinant...... chromatography procedure using the immobilized anti-uPAR monoclonal antibody R2. An optional enterokinase cleavage site is included between the various recombinant proteins and the linker region of the tag, which enables generation of highly pure preparations of tag-free recombinant proteins. Using this system...

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.

Determination of soluble protein contents from RVNRL

International Nuclear Information System (INIS)

Wan Manshol Wan Zin; Nurulhuda Othman

1996-01-01

This project was carried out to determine the soluble protein contents on RVNRL film vulcanisates, with respect to the RVNRL storage time, gamma irradiation dose absorbed by the latex and the effect of different leaching time and leaching conditions. These three factors are important in the hope to determine the best possible mean of minimizing the soluble protein contents in products made from RVNRL. Within the nine months storage period employed in the study, the results show that, the longer the storage period the less the soluble protein extracted from the film samples. Gamma irradiation dose absorbed by the samples, between 5.3 kGy to 25.2 kGy seems to influence the soluble protein contents of the RVNRL films vulcanisates. The higher the dose the more was the soluble protein extracted from the film samples. At an absorbed dose of 5.3 kGy and 25.2 kGy, the soluble contents were 0. 198 mg/ml and 0.247 mg/ml respectively. At a fixed leaching temperature, the soluble proteins increases with leaching time and at a fixed leaching time, the soluble proteins increases with leaching temperature. ne highest extractable protein contents was determined at a leaching time of 10 minutes and leaching temperature of 90'C The protein analysis were done by using Modified Lowry Method

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.

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

Toxoplasma gondii: biochemical and biophysical characterization of recombinant soluble dense granule proteins GRA2 and GRA6.

Science.gov (United States)

Bittame, Amina; Effantin, Grégory; Pètre, Graciane; Ruffiot, Pauline; Travier, Laetitia; Schoehn, Guy; Weissenhorn, Winfried; Cesbron-Delauw, Marie-France; Gagnon, Jean; Mercier, Corinne

2015-03-27

The most prominent structural feature of the parasitophorous vacuole (PV) in which the intracellular parasite Toxoplasma gondii proliferates is a membranous nanotubular network (MNN), which interconnects the parasites and the PV membrane. The MNN function remains unclear. The GRA2 and GRA6 proteins secreted from the parasite dense granules into the PV have been implicated in the MNN biogenesis. Amphipathic alpha-helices (AAHs) predicted in GRA2 and an alpha-helical hydrophobic domain predicted in GRA6 have been proposed to be responsible for their membrane association, thereby potentially molding the MMN in its structure. Here we report an analysis of the recombinant proteins (expressed in detergent-free conditions) by circular dichroism, which showed that full length GRA2 displays an alpha-helical secondary structure while recombinant GRA6 and GRA2 truncated of its AAHs are mainly random coiled. Dynamic light scattering and transmission electron microscopy showed that recombinant GRA6 and truncated GRA2 constitute a homogenous population of small particles (6-8 nm in diameter) while recombinant GRA2 corresponds to 2 populations of particles (∼8-15 nm and up to 40 nm in diameter, respectively). The unusual properties of GRA2 due to its AAHs are discussed. Copyright © 2015 Elsevier Inc. All rights reserved.

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.

Expression and purification of recombinant hemoglobin in Escherichia coli

DEFF Research Database (Denmark)

Natarajan, Chandrasekhar; Jiang, Xiaoben; Fago, Angela

2011-01-01

BACKGROUND: Recombinant DNA technologies have played a pivotal role in the elucidation of structure-function relationships in hemoglobin (Hb) and other globin proteins. Here we describe the development of a plasmid expression system to synthesize recombinant Hbs in Escherichia coli, and we describe...... a protocol for expressing Hbs with low intrinsic solubilities. Since the alpha- and beta-chain Hbs of different species span a broad range of solubilities, experimental protocols that have been optimized for expressing recombinant human HbA may often prove unsuitable for the recombinant expression......-translational modifications. CONCLUSION/SIGNIFICANCE: Our protocol should prove useful for the experimental study of recombinant Hbs in many non-human animals. One of the chief advantages of our protocol is that we can express soluble recombinant Hb without co-expressing molecular chaperones, and without the need...

Maltose-binding protein enhances secretion of recombinant human granzyme B accompanied by in vivo processing of a precursor MBP fusion protein.

Directory of Open Access Journals (Sweden)

Benjamin Dälken

Full Text Available BACKGROUND: The apoptosis-inducing serine protease granzyme B (GrB is an important factor contributing to lysis of target cells by cytotoxic lymphocytes. Expression of enzymatically active GrB in recombinant form is a prerequisite for functional analysis and application of GrB for therapeutic purposes. METHODS AND FINDINGS: We investigated the influence of bacterial maltose-binding protein (MBP fused to GrB via a synthetic furin recognition motif on the expression of the MBP fusion protein also containing an N-terminal α-factor signal peptide in the yeast Pichia pastoris. MBP markedly enhanced the amount of GrB secreted into culture supernatant, which was not the case when GrB was fused to GST. MBP-GrB fusion protein was cleaved during secretion by an endogenous furin-like proteolytic activity in vivo, liberating enzymatically active GrB without the need of subsequent in vitro processing. Similar results were obtained upon expression of a recombinant fragment of the ErbB2/HER2 receptor protein or GST as MBP fusions. CONCLUSIONS: Our results demonstrate that combination of MBP as a solubility enhancer with specific in vivo cleavage augments secretion of processed and functionally active proteins from yeast. This strategy may be generally applicable to improve folding and increase yields of recombinant proteins.

Maltose-Binding Protein Enhances Secretion of Recombinant Human Granzyme B Accompanied by In Vivo Processing of a Precursor MBP Fusion Protein

Science.gov (United States)

Dälken, Benjamin; Jabulowsky, Robert A.; Oberoi, Pranav; Benhar, Itai; Wels, Winfried S.

2010-01-01

Background The apoptosis-inducing serine protease granzyme B (GrB) is an important factor contributing to lysis of target cells by cytotoxic lymphocytes. Expression of enzymatically active GrB in recombinant form is a prerequisite for functional analysis and application of GrB for therapeutic purposes. Methods and Findings We investigated the influence of bacterial maltose-binding protein (MBP) fused to GrB via a synthetic furin recognition motif on the expression of the MBP fusion protein also containing an N-terminal α-factor signal peptide in the yeast Pichia pastoris. MBP markedly enhanced the amount of GrB secreted into culture supernatant, which was not the case when GrB was fused to GST. MBP-GrB fusion protein was cleaved during secretion by an endogenous furin-like proteolytic activity in vivo, liberating enzymatically active GrB without the need of subsequent in vitro processing. Similar results were obtained upon expression of a recombinant fragment of the ErbB2/HER2 receptor protein or GST as MBP fusions. Conclusions Our results demonstrate that combination of MBP as a solubility enhancer with specific in vivo cleavage augments secretion of processed and functionally active proteins from yeast. This strategy may be generally applicable to improve folding and increase yields of recombinant proteins. PMID:21203542

Recombinant protein expression of Moringa oleifera lectin in methylotrophic yeast as active coagulant for sustainable high turbid water treatment.

Science.gov (United States)

Abd Wahid, Muhamad Azhar; Megat Mohd Noor, Megat Johari; Goto, Masafumi; Sugiura, Norio; Othman, Nor'azizi; Zakaria, Zuriati; Ahmad Mohammed, Thamer; Jusoh, Ahmad; Hara, Hirofumi

2017-08-01

The natural coagulant Moringa oleifera lectin (MoL) as cationic protein is a promising candidate in coagulation process of water treatment plant. Introducing the gene encoding MoL into a host, Pichia pastoris, to secrete soluble recombinant protein is assessed in this study. Initial screening using PCR confirmed the insertion of MoL gene, and SDS-PAGE analysis detected the MoL protein at 8 kDa. Cultured optimization showed the highest MoL protein at 520 mg/L was observed at 28 °C for 144 h of culturing by induction in 1% methanol. Approximately, 0.40 mg/mL of recombinant MoL protein showed 95 ± 2% turbidity removal of 1% kaolin suspension. In 0.1% kaolin suspension, the concentration of MoL at 10 μg/mL exhibits the highest turbidity reduction at 68 ± 1%. Thus, recombinant MoL protein from P. pastoris is an effective coagulant for water treatment.

Expression and purification of recombinant hemoglobin in Escherichia coli.

Directory of Open Access Journals (Sweden)

Chandrasekhar Natarajan

Full Text Available Recombinant DNA technologies have played a pivotal role in the elucidation of structure-function relationships in hemoglobin (Hb and other globin proteins. Here we describe the development of a plasmid expression system to synthesize recombinant Hbs in Escherichia coli, and we describe a protocol for expressing Hbs with low intrinsic solubilities. Since the α- and β-chain Hbs of different species span a broad range of solubilities, experimental protocols that have been optimized for expressing recombinant human HbA may often prove unsuitable for the recombinant expression of wildtype and mutant Hbs of other species.As a test case for our expression system, we produced recombinant Hbs of the deer mouse (Peromyscus maniculatus, a species that has been the subject of research on mechanisms of Hb adaptation to hypoxia. By experimentally assessing the combined effects of induction temperature, induction time and E. coli expression strain on the solubility of recombinant deer mouse Hbs, we identified combinations of expression conditions that greatly enhanced the yield of recombinant protein and which also increased the efficiency of post-translational modifications.Our protocol should prove useful for the experimental study of recombinant Hbs in many non-human animals. One of the chief advantages of our protocol is that we can express soluble recombinant Hb without co-expressing molecular chaperones, and without the need for additional reconstitution or heme-incorporation steps. Moreover, our plasmid construct contains a combination of unique restriction sites that allows us to produce recombinant Hbs with different α- and β-chain subunit combinations by means of cassette mutagenesis.

Expression and purification of recombinant hemoglobin in Escherichia coli.

Science.gov (United States)

Natarajan, Chandrasekhar; Jiang, Xiaoben; Fago, Angela; Weber, Roy E; Moriyama, Hideaki; Storz, Jay F

2011-01-01

Recombinant DNA technologies have played a pivotal role in the elucidation of structure-function relationships in hemoglobin (Hb) and other globin proteins. Here we describe the development of a plasmid expression system to synthesize recombinant Hbs in Escherichia coli, and we describe a protocol for expressing Hbs with low intrinsic solubilities. Since the α- and β-chain Hbs of different species span a broad range of solubilities, experimental protocols that have been optimized for expressing recombinant human HbA may often prove unsuitable for the recombinant expression of wildtype and mutant Hbs of other species. As a test case for our expression system, we produced recombinant Hbs of the deer mouse (Peromyscus maniculatus), a species that has been the subject of research on mechanisms of Hb adaptation to hypoxia. By experimentally assessing the combined effects of induction temperature, induction time and E. coli expression strain on the solubility of recombinant deer mouse Hbs, we identified combinations of expression conditions that greatly enhanced the yield of recombinant protein and which also increased the efficiency of post-translational modifications. Our protocol should prove useful for the experimental study of recombinant Hbs in many non-human animals. One of the chief advantages of our protocol is that we can express soluble recombinant Hb without co-expressing molecular chaperones, and without the need for additional reconstitution or heme-incorporation steps. Moreover, our plasmid construct contains a combination of unique restriction sites that allows us to produce recombinant Hbs with different α- and β-chain subunit combinations by means of cassette mutagenesis.

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.

Comparing side chain packing in soluble proteins, protein-protein interfaces, and transmembrane proteins.

Science.gov (United States)

Gaines, J C; Acebes, S; Virrueta, A; Butler, M; Regan, L; O'Hern, C S

2018-05-01

We compare side chain prediction and packing of core and non-core regions of soluble proteins, protein-protein interfaces, and transmembrane proteins. We first identified or created comparable databases of high-resolution crystal structures of these 3 protein classes. We show that the solvent-inaccessible cores of the 3 classes of proteins are equally densely packed. As a result, the side chains of core residues at protein-protein interfaces and in the membrane-exposed regions of transmembrane proteins can be predicted by the hard-sphere plus stereochemical constraint model with the same high prediction accuracies (>90%) as core residues in soluble proteins. We also find that for all 3 classes of proteins, as one moves away from the solvent-inaccessible core, the packing fraction decreases as the solvent accessibility increases. However, the side chain predictability remains high (80% within 30°) up to a relative solvent accessibility, rSASA≲0.3, for all 3 protein classes. Our results show that ≈40% of the interface regions in protein complexes are "core", that is, densely packed with side chain conformations that can be accurately predicted using the hard-sphere model. We propose packing fraction as a metric that can be used to distinguish real protein-protein interactions from designed, non-binding, decoys. Our results also show that cores of membrane proteins are the same as cores of soluble proteins. Thus, the computational methods we are developing for the analysis of the effect of hydrophobic core mutations in soluble proteins will be equally applicable to analyses of mutations in membrane proteins. © 2018 Wiley Periodicals, Inc.

ESPRIT: an automated, library-based method for mapping and soluble expression of protein domains from challenging targets.

Science.gov (United States)

Yumerefendi, Hayretin; Tarendeau, Franck; Mas, Philippe J; Hart, Darren J

2010-10-01

Expression of sufficient quantities of soluble protein for structural biology and other applications is often a very difficult task, especially when multimilligram quantities are required. In order to improve yield, solubility or crystallisability of a protein, it is common to subclone shorter genetic constructs corresponding to single- or multi-domain fragments. However, it is not always clear where domain boundaries are located, especially when working on novel targets with little or no sequence similarity to other proteins. Several methods have been described employing aspects of directed evolution to the recombinant expression of challenging proteins. These combine the construction of a random library of genetic constructs of a target with a screening or selection process to identify solubly expressing protein fragments. Here we review several datasets from the ESPRIT (Expression of Soluble Proteins by Random Incremental Truncation) technology to provide a view on its capabilities. Firstly, we demonstrate how it functions using the well-characterised NF-kappaB p50 transcription factor as a model system. Secondly, application of ESPRIT to the challenging PB2 subunit of influenza polymerase has led to several novel atomic resolution structures; here we present an overview of the screening phase of that project. Thirdly, analysis of the human kinase TBK1 is presented to show how the ESPRIT technology rapidly addresses the compatibility of challenging targets with the Escherichia coli expression system.

Evaluation of a competitive enzyme-linked immunosorbent assay for measurements of soluble HLA-G protein.

Science.gov (United States)

Rasmussen, M; Dahl, M; Buus, S; Djurisic, S; Ohlsson, J; Hviid, T V F

2014-08-01

The human leukocyte antigen (HLA) class Ib molecule, HLA-G, has gained increased attention because of its assumed important role in immune regulation. The HLA-G protein exists in several soluble isoforms. Most important are the actively secreted HLA-G5 full-length isoform generated by alternative splicing retaining intron 4 with a premature stop codon, and the cleavage of full-length membrane-bound HLA-G1 from the cell surface, so-called soluble HLA-G1 (sHLA-G1). A specific and sensitive immunoassay for measurements of soluble HLA-G is mandatory for conceivable routine testing and research projects. We report a novel method, a competitive immunoassay, for measuring HLA-G5/sHLA-G1 in biological fluids. The sHLA-G immunoassay is based upon a competitive enzyme-linked immunosorbent assay (ELISA) principle. It includes a recombinant sHLA-G1 protein in complex with β2-microglobulin and a peptide as a standard, biotinylated recombinant sHLA-G1 as an indicator, and the MEM-G/9 anti-HLA-G monoclonal antibody (mAb) as the capture antibody. The specificity and sensitivity of the assay were evaluated. Testing with different recombinant HLA class I proteins and different anti-HLA class I mAbs showed that the sHLA-G immunoassay was highly specific. Optimal combinations of competitor sHLA-G1 and capture mAb concentrations were determined. Two versions of the assay were tested. One with a relatively wide dynamic range from 3.1 to 100.0 ng/ml, and another more sensitive version ranging from 1.6 to 12.5 ng/ml. An intra-assay coefficient of variation (CV) of 15.5% at 88 ng/ml and an inter-assay CV of 23.1% at 39 ng/ml were determined. An assay based on the competitive sHLA-G ELISA may be important for measurements of sHLA-G proteins in several conditions: assisted reproduction, organ transplantation, cancer, and certain pregnancy complications, both in research studies and possibly in the future also for clinical routine use. © 2014 John Wiley & Sons A/S. Published by John Wiley

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

Comparative study to develop a single method for retrieving wide class of recombinant proteins from classical inclusion bodies.

Science.gov (United States)

Padhiar, Arshad Ahmed; Chanda, Warren; Joseph, Thomson Patrick; Guo, Xuefang; Liu, Min; Sha, Li; Batool, Samana; Gao, Yifan; Zhang, Wei; Huang, Min; Zhong, Mintao

2018-03-01

The formation of inclusion bodies (IBs) is considered as an Achilles heel of heterologous protein expression in bacterial hosts. Wide array of techniques has been developed to recover biochemically challenging proteins from IBs. However, acquiring the active state even from the same protein family was found to be an independent of single established method. Here, we present a new strategy for the recovery of wide sub-classes of recombinant protein from harsh IBs. We found that numerous methods and their combinations for reducing IB formation and producing soluble proteins were not effective, if the inclusion bodies were harsh in nature. On the other hand, different practices with mild solubilization buffers were able to solubilize IBs completely, yet the recovery of active protein requires large screening of refolding buffers. With the integration of previously reported mild solubilization techniques, we proposed an improved method, which comprised low sarkosyl concentration, ranging from 0.05 to 0.1% coupled with slow freezing (- 1 °C/min) and fast thaw (room temperature), resulting in greater solubility and the integrity of solubilized protein. Dilution method was employed with single buffer to restore activity for every sub-class of recombinant protein. Results showed that the recovered protein's activity was significantly higher compared with traditional solubilization/refolding approach. Solubilization of IBs by the described method was proved milder in nature, which restored native-like conformation of proteins within IBs.

Soluble cysteine-rich tick saliva proteins Salp15 and Iric-1 from E. coli.

Science.gov (United States)

Kolb, Philipp; Vorreiter, Jolanta; Habicht, Jüri; Bentrop, Detlef; Wallich, Reinhard; Nassal, Michael

2015-01-01

Ticks transmit numerous pathogens, including borreliae, which cause Lyme disease. Tick saliva contains a complex mix of anti-host defense factors, including the immunosuppressive cysteine-rich secretory glycoprotein Salp15 from Ixodes scapularis ticks and orthologs like Iric-1 from Ixodes ricinus. All tick-borne microbes benefit from the immunosuppression at the tick bite site; in addition, borreliae exploit the binding of Salp15 to their outer surface protein C (OspC) for enhanced transmission. Hence, Salp15 proteins are attractive targets for anti-tick vaccines that also target borreliae. However, recombinant Salp proteins are not accessible in sufficient quantity for either vaccine manufacturing or for structural characterization. As an alternative to low-yield eukaryotic systems, we investigated cytoplasmic expression in Escherichia coli, even though this would not result in glycosylation. His-tagged Salp15 was efficiently expressed but insoluble. Among the various solubility-enhancing protein tags tested, DsbA was superior, yielding milligram amounts of soluble, monomeric Salp15 and Iric-1 fusions. Easily accessible mutants enabled epitope mapping of two monoclonal antibodies that, importantly, cross-react with glycosylated Salp15, and revealed interaction sites with OspC. Free Salp15 and Iric-1 from protease-cleavable fusions, despite limited solubility, allowed the recording of (1)H-(15)N 2D NMR spectra, suggesting partial folding of the wild-type proteins but not of Cys-free variants. Fusion to the NMR-compatible GB1 domain sufficiently enhanced solubility to reveal first secondary structure elements in (13)C/(15)N double-labeled Iric-1. Together, E. coli expression of appropriately fused Salp15 proteins may be highly valuable for the molecular characterization of the function and eventually the 3D structure of these medically relevant tick proteins.

Recombinant expression and purification of the RNA-binding LARP6 proteins from fish genetic model organisms.

Science.gov (United States)

Castro, José M; Horn, Daniel A; Pu, Xinzhu; Lewis, Karen A

2017-06-01

The RNA-binding proteins that comprise the La-related protein (LARP) superfamily have been implicated in a wide range of cellular functions, from tRNA maturation to regulation of protein synthesis. To more expansively characterize the biological function of the LARP6 subfamily, we have recombinantly expressed the full-length LARP6 proteins from two teleost fish, platyfish (Xiphophorus maculatus) and zebrafish (Danio rerio). The yields of the recombinant proteins were enhanced to >2 mg/L using a tandem approach of an N-terminal His 6 -SUMO tag and an iterative solubility screening assay to identify structurally stabilizing buffer components. The domain topologies of the purified fish proteins were probed with limited proteolysis. The fish proteins contain an internal, protease-resistant 40 kDa domain, which is considerably more stable than the comparable domain from the human LARP6 protein. The fish proteins are therefore a lucrative model system in which to study both the evolutionary divergence of this family of La-related proteins and the structure and conformational dynamics of the domains that comprise the LARP6 protein. Copyright © 2017 Elsevier Inc. All rights reserved.

Prokaryotic Soluble Overexpression and Purification of Human VEGF165 by Fusion to a Maltose Binding Protein Tag.

Directory of Open Access Journals (Sweden)

Minh Tan Nguyen

Full Text Available Human vascular endothelial growth factor (VEGF is a key regulator of angiogenesis and plays a central role in the process of tumor growth and metastatic dissemination. Escherichia coli is one of the most common expression systems used for the production of recombinant proteins; however, expression of human VEGF in E. coli has proven difficult because the E. coli-expressed VEGF tends to be misfolded and forms inclusion bodies, resulting in poor solubility. In this study, we successfully produced semi-preparative amounts of soluble bioactive human VEGF165 (hVEGF. We created seven N-terminal fusion tag constructs with hexahistidine (His6, thioredoxin (Trx, glutathione S-transferase (GST, maltose-binding protein (MBP, N-utilization substance protein A (NusA, human protein disulfide isomerase (PDI, and the b'a' domain of PDI (PDIb'a', and tested each construct for soluble overexpression in E. coli. We found that at 18°C, 92.8% of the MBP-tagged hVEGF to be soluble and that this tag significantly increased the protein's solubility. We successfully purified 0.8 mg of pure hVEGF per 500 mL cell culture. The purified hVEGF is stable after tag cleavage, contains very low levels of endotoxin, and is 97.6% pure. Using an Flk1+ mesodermal precursor cell (MPC differentiation assay, we show that the purified hVEGF is not only bioactive but has similar bioactivity to hVEGF produced in mammalian cells. Previous reports on producing hVEGF in E. coli have all been based on refolding of the protein from inclusion bodies. To our knowledge, this is the first report on successfully expressing and purifying soluble hVEGF in E. coli.

X-radiation effect on soluble proteins of gastric mucosa

International Nuclear Information System (INIS)

Sukhomlinov, B.F.; Chajka, Ya.P.; Fedorovich, A.N.

1979-01-01

Using the method of electrophoresis in agar gel soluble proteins of gastric mucosa of rats were separated into 11 fractions. Proteins posessing a proteolytic (pH 1.8) and lipase (pH 7.4) activity were localized within the second and third prealbumin fractions. Soluble proteins of gastric mucosa contain glyco- and lipoproteid complexes. Exposure of rats to 1000 R of X-rays induces quantitative redistribution within the electrophoretic spectrum of soluble proteins and a considerable disturbance of the proteolytic activity of total soluble proteins throughout the entire period of observation (from 10 min to 72h)

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.

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.

Soluble expression and purification of the recombinant bioactive peptide precursor BPP-1 in Escherichia coli using a cELP-SUMO dual fusion system.

Science.gov (United States)

Rao, Shengqi; Zang, Xiangyu; Yang, Zhenquan; Gao, Lu; Yin, Yongqi; Fang, Weiming

2016-02-01

A bioactive peptide precursor (BPP-1, 14.3 kDa/115AA), a newly designed polypeptide that may exert a potential antihypertensive effect in vivo, is composed of many different ACE inhibitory peptides and antioxidant peptides tandemly linked according to the restriction sites of gastrointestinal proteases. In this report, we present a novel method to obtain soluble BPP-1 in Escherichia coli using cationic elastin-like polypeptide and SUMO (cELP-SUMO) tags. The cELP-SUMO-tagged fusion protein was expressed in soluble form at 20 °C for 20 h. After purification based on the inverse transition cycling (ITC) method, the purified cELP-SUMO-CFPP fusion protein was subsequently cleaved by a SUMO protease to release the mature BPP-1. After a subsequent simple salt precipitation process, approximately 167.2 mg of recombinant BPP-1 was obtained from 1 l of bacterial culture with at least 92% purity. The molecular mass (Mr) of the recombinant BPP-1 was confirmed by MALDI-TOF MS to equal 14,347. The purified BPP-1 was subjected to simulated gastrointestinal digestion, and the resulting hydrolysates exhibited notable ACE inhibitory and antioxidant activities in vitro. This report provides the first description of the soluble production of a bioactive peptide multimer with potential ACE inhibitory and antioxidant activities in E. coli using a cELP-SUMO tag. Copyright © 2015 Elsevier Inc. All rights reserved.

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.

Characterization of soluble protein BCP 11/24 from bovine corneal epithelium, different from the principal soluble protein BCP 54

NARCIS (Netherlands)

Bakker, C.; Pasmans, S.; Verhagen, C.; van Haren, M.; van der Gaag, R.; Hoekzema, R.

1992-01-01

The water-soluble fraction of bovine corneal epithelium was analysed by polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE). Next to the principal soluble protein BCP 54, which has recently been identified as a corneal aldehyde dehydrogenase (ALDH), another abundant protein was

Relationship between Molecular Structure Characteristics of Feed Proteins and Protein Digestibility and Solubility

Directory of Open Access Journals (Sweden)

Mingmei Bai

2016-08-01

Full Text Available The nutritional value of feed proteins and their utilization by livestock are related not only to the chemical composition but also to the structure of feed proteins, but few studies thus far have investigated the relationship between the structure of feed proteins and their solubility as well as digestibility in monogastric animals. To address this question we analyzed soybean meal, fish meal, corn distiller’s dried grains with solubles, corn gluten meal, and feather meal by Fourier transform infrared (FTIR spectroscopy to determine the protein molecular spectral band characteristics for amides I and II as well as α-helices and β-sheets and their ratios. Protein solubility and in vitro digestibility were measured with the Kjeldahl method using 0.2% KOH solution and the pepsin-pancreatin two-step enzymatic method, respectively. We found that all measured spectral band intensities (height and area of feed proteins were correlated with their the in vitro digestibility and solubility (p≤0.003; moreover, the relatively quantitative amounts of α-helices, random coils, and α-helix to β-sheet ratio in protein secondary structures were positively correlated with protein in vitro digestibility and solubility (p≤0.004. On the other hand, the percentage of β-sheet structures was negatively correlated with protein in vitro digestibility (p<0.001 and solubility (p = 0.002. These results demonstrate that the molecular structure characteristics of feed proteins are closely related to their in vitro digestibility at 28 h and solubility. Furthermore, the α-helix-to-β-sheet ratio can be used to predict the nutritional value of feed proteins.

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.

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.

Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production.

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Raheem Ullah

Full Text Available Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications.

Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production.

Science.gov (United States)

Ullah, Raheem; Shah, Majid Ali; Tufail, Soban; Ismat, Fouzia; Imran, Muhammad; Iqbal, Mazhar; Mirza, Osman; Rhaman, Moazur

2016-01-01

Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications.

Lysine Rich Proteins in the Salt-Soluble Protein Fraction of Barley

DEFF Research Database (Denmark)

Ingversen, J.; Køie, B.

1973-01-01

Fractionation of the protein complex from Emir barley showed that the salt-soluble fraction accounts for 44% of the total lysine content but only for 2.......Fractionation of the protein complex from Emir barley showed that the salt-soluble fraction accounts for 44% of the total lysine content but only for 2....

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....

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

A SEP tag enhances the expression, solubility and yield of recombinant TEV protease without altering its activity.

Science.gov (United States)

Nautiyal, Kalpana; Kuroda, Yutaka

2018-05-25

Tobacco Etch Virus (TEV) protease is used in the purification of recombinant proteins, but its usage is often hampered by solubility issues. Here, we report a short, 12-residue solubility enhancing peptide (SEP) tag attached at the C-terminus of TEV (TEV-C9R). We assessed the effects of the C9R tag on the biophysical and biochemical characteristics of TEV. The yield of HPLC purified TEV-C9R expressed in E. coli grown in 200 mL LB or TB media was between 10 and 13 mg, which was up to 6.5 times higher than the yield of the untagged TEV (untagged-TEV). TEV-C9R was active over a pH range of 5-8, which was wider than that of the commonly used thrombin, and it remained active upon incubation at 60 °C much longer than the untagged-TEV, which aggregated at this temperature. Static and dynamic light scattering demonstrated the higher solubility of purified TEV-C9R. Furthermore, the thermal unfolding of TEV-C9R, as assessed by circular dichroism at pH 4.7, was almost perfectly reversible, in contrast to that of untagged-TEV, which aggregated at high temperature. These results demonstrate the improved biophysical and biochemical characteristics of TEV-C9R originating from higher solubility and provide another example of how SEP tags can enhance enzyme solubility without altering its activity. Copyright © 2018 Elsevier B.V. All rights reserved.

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 ...

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.

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.

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....

The Induction of Recombinant Protein Bodies in Different Subcellular Compartments Reveals a Cryptic Plastid-Targeting Signal in the 27-kDa γ-Zein Sequence

Energy Technology Data Exchange (ETDEWEB)

Hofbauer, Anna; Peters, Jenny; Arcalis, Elsa [Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna (Austria); Rademacher, Thomas [Institute of Molecular Biotechnology, RWTH Aachen University, Aachen (Germany); Lampel, Johannes [Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna (Austria); Eudes, François [Agriculture and Agri-Food Canada, Lethbridge, AB (Canada); Vitale, Alessandro [Institute of Agricultural Biology and Biotechnology, National Research Council (CNR), Milan (Italy); Stoger, Eva, E-mail: eva.stoger@boku.ac.at [Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna (Austria)

2014-12-11

Naturally occurring storage proteins such as zeins are used as fusion partners for recombinant proteins because they induce the formation of ectopic storage organelles known as protein bodies (PBs) where the proteins are stabilized by intermolecular interactions and the formation of disulfide bonds. Endogenous PBs are derived from the endoplasmic reticulum (ER). Here, we have used different targeting sequences to determine whether ectopic PBs composed of the N-terminal portion of mature 27 kDa γ-zein added to a fluorescent protein could be induced to form elsewhere in the cell. The addition of a transit peptide for targeting to plastids causes PB formation in the stroma, whereas in the absence of any added targeting sequence PBs were typically associated with the plastid envelope, revealing the presence of a cryptic plastid-targeting signal within the γ-zein cysteine-rich domain. The subcellular localization of the PBs influences their morphology and the solubility of the stored recombinant fusion protein. Our results indicate that the biogenesis and budding of PBs does not require ER-specific factors and therefore, confirm that γ-zein is a versatile fusion partner for recombinant proteins offering unique opportunities for the accumulation and bioencapsulation of recombinant proteins in different subcellular compartments.

The Induction of Recombinant Protein Bodies in Different Subcellular Compartments Reveals a Cryptic Plastid-Targeting Signal in the 27-kDa γ-Zein Sequence

International Nuclear Information System (INIS)

Hofbauer, Anna; Peters, Jenny; Arcalis, Elsa; Rademacher, Thomas; Lampel, Johannes; Eudes, François; Vitale, Alessandro; Stoger, Eva

2014-01-01

Naturally occurring storage proteins such as zeins are used as fusion partners for recombinant proteins because they induce the formation of ectopic storage organelles known as protein bodies (PBs) where the proteins are stabilized by intermolecular interactions and the formation of disulfide bonds. Endogenous PBs are derived from the endoplasmic reticulum (ER). Here, we have used different targeting sequences to determine whether ectopic PBs composed of the N-terminal portion of mature 27 kDa γ-zein added to a fluorescent protein could be induced to form elsewhere in the cell. The addition of a transit peptide for targeting to plastids causes PB formation in the stroma, whereas in the absence of any added targeting sequence PBs were typically associated with the plastid envelope, revealing the presence of a cryptic plastid-targeting signal within the γ-zein cysteine-rich domain. The subcellular localization of the PBs influences their morphology and the solubility of the stored recombinant fusion protein. Our results indicate that the biogenesis and budding of PBs does not require ER-specific factors and therefore, confirm that γ-zein is a versatile fusion partner for recombinant proteins offering unique opportunities for the accumulation and bioencapsulation of recombinant proteins in different subcellular compartments.

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.

Two novel heat-soluble protein families abundantly expressed in an anhydrobiotic tardigrade.

Directory of Open Access Journals (Sweden)

Ayami Yamaguchi

Full Text Available Tardigrades are able to tolerate almost complete dehydration by reversibly switching to an ametabolic state. This ability is called anhydrobiosis. In the anhydrobiotic state, tardigrades can withstand various extreme environments including space, but their molecular basis remains largely unknown. Late embryogenesis abundant (LEA proteins are heat-soluble proteins and can prevent protein-aggregation in dehydrated conditions in other anhydrobiotic organisms, but their relevance to tardigrade anhydrobiosis is not clarified. In this study, we focused on the heat-soluble property characteristic of LEA proteins and conducted heat-soluble proteomics using an anhydrobiotic tardigrade. Our heat-soluble proteomics identified five abundant heat-soluble proteins. All of them showed no sequence similarity with LEA proteins and formed two novel protein families with distinct subcellular localizations. We named them Cytoplasmic Abundant Heat Soluble (CAHS and Secretory Abundant Heat Soluble (SAHS protein families, according to their localization. Both protein families were conserved among tardigrades, but not found in other phyla. Although CAHS protein was intrinsically unstructured and SAHS protein was rich in β-structure in the hydrated condition, proteins in both families changed their conformation to an α-helical structure in water-deficient conditions as LEA proteins do. Two conserved repeats of 19-mer motifs in CAHS proteins were capable to form amphiphilic stripes in α-helices, suggesting their roles as molecular shield in water-deficient condition, though charge distribution pattern in α-helices were different between CAHS and LEA proteins. Tardigrades might have evolved novel protein families with a heat-soluble property and this study revealed a novel repertoire of major heat-soluble proteins in these anhydrobiotic animals.

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.

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

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.

Relationship between Molecular Structure Characteristics of Feed Proteins and Protein In vitro Digestibility and Solubility.

Science.gov (United States)

Bai, Mingmei; Qin, Guixin; Sun, Zewei; Long, Guohui

2016-08-01

The nutritional value of feed proteins and their utilization by livestock are related not only to the chemical composition but also to the structure of feed proteins, but few studies thus far have investigated the relationship between the structure of feed proteins and their solubility as well as digestibility in monogastric animals. To address this question we analyzed soybean meal, fish meal, corn distiller's dried grains with solubles, corn gluten meal, and feather meal by Fourier transform infrared (FTIR) spectroscopy to determine the protein molecular spectral band characteristics for amides I and II as well as α-helices and β-sheets and their ratios. Protein solubility and in vitro digestibility were measured with the Kjeldahl method using 0.2% KOH solution and the pepsin-pancreatin two-step enzymatic method, respectively. We found that all measured spectral band intensities (height and area) of feed proteins were correlated with their the in vitro digestibility and solubility (p≤0.003); moreover, the relatively quantitative amounts of α-helices, random coils, and α-helix to β-sheet ratio in protein secondary structures were positively correlated with protein in vitro digestibility and solubility (p≤0.004). On the other hand, the percentage of β-sheet structures was negatively correlated with protein in vitro digestibility (pdigestibility at 28 h and solubility. Furthermore, the α-helix-to-β-sheet ratio can be used to predict the nutritional value of feed proteins.

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

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.

Unique Features of Halophilic Proteins.

Science.gov (United States)

Arakawa, Tsutomu; Yamaguchi, Rui; Tokunaga, Hiroko; Tokunaga, Masao

2017-01-01

Proteins from moderate and extreme halophiles have unique characteristics. They are highly acidic and hydrophilic, similar to intrinsically disordered proteins. These characteristics make the halophilic proteins soluble in water and fold reversibly. In addition to reversible folding, the rate of refolding of halophilic proteins from denatured structure is generally slow, often taking several days, for example, for extremely halophilic proteins. This slow folding rate makes the halophilic proteins a novel model system for folding mechanism analysis. High solubility and reversible folding also make the halophilic proteins excellent fusion partners for soluble expression of recombinant proteins.

Facilitating protein solubility by use of peptide extensions

Science.gov (United States)

Freimuth, Paul I; Zhang, Yian-Biao; Howitt, Jason

2013-09-17

Expression vectors for expression of a protein or polypeptide of interest as a fusion product composed of the protein or polypeptide of interest fused at one terminus to a solubility enhancing peptide extension are provided. Sequences encoding the peptide extensions are provided. The invention further comprises antibodies which bind specifically to one or more of the solubility enhancing peptide extensions.

Recombinant HT{sub m4} gene, protein and assays

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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.

EFFECT OF HEAT TREATMENT ON SOYBEAN PROTEIN SOLUBILITY

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RODICA CĂPRIŢĂ

2007-05-01

Full Text Available The use of soybean products in animal feeds is limited due to the presence of antinutritional factors (ANF. Proper heat processing is required to destroy ANF naturally present in raw soybeans and to remove solvent remaining from the oil extraction process. Over and under toasting of soybean causes lower nutritional value. Excessive heat treatment causes Maillard reaction which affects the availability of lysine in particular and produces changes to the chemical structure of proteins resulting in a decrease of the nutritive value. The objective of this study was to evaluate the effect of heating time on the protein solubility. The investigation of the heating time on protein solubility in soybean meal (SBM revealed a negative correlation (r = -0.9596. Since the urease index is suitable only for detecting under processed SBM, the protein solubility is an important index for monitoring SBM quality.

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.

Self assembling nanocomposites for protein delivery: supramolecular interactions of soluble polymers with protein drugs.

Science.gov (United States)

Salmaso, Stefano; Caliceti, Paolo

2013-01-02

Translation of therapeutic proteins to pharmaceutical products is often encumbered by their inadequate physicochemical and biopharmaceutical properties, namely low stability and poor bioavailability. Over the last decades, several academic and industrial research programs have been focused on development of biocompatible polymers to produce appropriate formulations that provide for enhanced therapeutic performance. According to their physicochemical properties, polymers have been exploited to obtain a variety of formulations including biodegradable microparticles, 3-dimensional hydrogels, bioconjugates and soluble nanocomposites. Several soluble polymers bearing charges or hydrophobic moieties along the macromolecular backbone have been found to physically associate with proteins to form soluble nanocomplexes. Physical complexation is deemed a valuable alternative tool to the chemical bioconjugation. Soluble protein/polymer nanocomplexes formed by physical specific or unspecific interactions have been found in fact to possess peculiar physicochemical, and biopharmaceutical properties. Accordingly, soluble polymeric systems have been developed to increase the protein stability, enhance the bioavailability, promote the absorption across the biological barriers, and prolong the protein residence in the bloodstream. Furthermore, a few polymers have been found to favour the protein internalisation into cells or boost their immunogenic potential by acting as immunoadjuvant in vaccination protocols. Copyright © 2011 Elsevier B.V. All rights reserved.

Membrane Proteins Are Dramatically Less Conserved than Water-Soluble Proteins across the Tree of Life.

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Sojo, Victor; Dessimoz, Christophe; Pomiankowski, Andrew; Lane, Nick

2016-11-01

Membrane proteins are crucial in transport, signaling, bioenergetics, catalysis, and as drug targets. Here, we show that membrane proteins have dramatically fewer detectable orthologs than water-soluble proteins, less than half in most species analyzed. This sparse distribution could reflect rapid divergence or gene loss. We find that both mechanisms operate. First, membrane proteins evolve faster than water-soluble proteins, particularly in their exterior-facing portions. Second, we demonstrate that predicted ancestral membrane proteins are preferentially lost compared with water-soluble proteins in closely related species of archaea and bacteria. These patterns are consistent across the whole tree of life, and in each of the three domains of archaea, bacteria, and eukaryotes. Our findings point to a fundamental evolutionary principle: membrane proteins evolve faster due to stronger adaptive selection in changing environments, whereas cytosolic proteins are under more stringent purifying selection in the homeostatic interior of the cell. This effect should be strongest in prokaryotes, weaker in unicellular eukaryotes (with intracellular membranes), and weakest in multicellular eukaryotes (with extracellular homeostasis). We demonstrate that this is indeed the case. Similarly, we show that extracellular water-soluble proteins exhibit an even stronger pattern of low homology than membrane proteins. These striking differences in conservation of membrane proteins versus water-soluble proteins have important implications for evolution and medicine. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Relationship between recombinant protein expression and host metabolome as determined by two-dimensional NMR spectroscopy.

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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.

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...

Manufacturing of recombinant therapeutic proteins in microbial systems.

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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.

Viral vectors for production of recombinant proteins in plants.

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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.

Identification of the hemoglobin scavenger receptor/CD163 as a natural soluble protein in plasma

DEFF Research Database (Denmark)

Møller, Holger Jon; Peterslund, Niels Anker; Graversen, Jonas Heilskov

2002-01-01

enabled identification of a soluble plasma form of HbSR (sHbSR) having an electrophoretic mobility equal to that of recombinant HbSR consisting of the extracellular domain (scavenger receptor cysteine-rich 1-9). A sandwich enzyme-linked immunosorbent assay was established and used to measure the s...... a level of sHbSR above the range of healthy persons. Patients with myelomonocytic leukemias and pneumonia/sepsis exhibited the highest levels (up to 67.3 mg/L). In conclusion, sHbSR is an abundant plasma protein potentially valuable in monitoring patients with infections and myelomonocytic leukemia....

A Soluble, Folded Protein without Charged Amino Acid Residues

DEFF Research Database (Denmark)

Højgaard, Casper; Kofoed, Christian; Espersen, Roall

2016-01-01

Charges are considered an integral part of protein structure and function, enhancing solubility and providing specificity in molecular interactions. We wished to investigate whether charged amino acids are indeed required for protein biogenesis and whether a protein completely free of titratable...... side chains can maintain solubility, stability, and function. As a model, we used a cellulose-binding domain from Cellulomonas fimi, which, among proteins of more than 100 amino acids, presently is the least charged in the Protein Data Bank, with a total of only four titratable residues. We find...

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...

Design and production of various fusion proteins of the nicotinamide/nicotinate mononucleotide adenilil transferase (NMNAT of Plasmodium falciparum

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Carlos Alfonso Nieto Clavijo

2017-09-01

Full Text Available Recombinant proteins have become useful tools in biochemistry research. During their production, however, inclusion bodies (IB appear, on the one hand, due to the high expression rate from the recombinant plasmids, which have high efficiency promoters, and, on the other hand, intrinsic characteristics of the expressed protein. Furhtermore, the nicotinamide/nicotinate mononucleotide adenilyl transferase (NMNAT is a central protein in NAD(H+ biosynthesis, an essential cofactor in cell metabolism, and in protozoon parasite has been studied. To study the NMNAT protein of these parasites, their recombinant version in E. coli has been expressed, getting a great quantity of IB as a by-product. To increase the solubility of the protein, the coding sequence of the NMNAT enzyme of Plasmodium falciparum was cloned in different expression plasmids which were subsequently transformed into E. coli BL21(DE3 expression strain. The solubility of the recombinant proteins was assessed and the one with the highest presence in the soluble fraction was subsequently purified and its enzyme activity was determined. The recombinant protein with a MBP (maltose-binding protein tag showed an increased solubility and purity.

Synthesis and characterization of recombinant abductin-based proteins.

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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.

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

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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.

Tryptophan tags and de novo designed complementary affinity ligands for the expression and purification of recombinant proteins.

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Pina, Ana Sofia; Carvalho, Sara; Dias, Ana Margarida G C; Guilherme, Márcia; Pereira, Alice S; Caraça, Luciana T; Coroadinha, Ana Sofia; Lowe, Christopher R; Roque, A Cecília A

2016-11-11

A common strategy for the production and purification of recombinant proteins is to fuse a tag to the protein terminal residues and employ a "tag-specific" ligand for fusion protein capture and purification. In this work, we explored the effect of two tryptophan-based tags, NWNWNW and WFWFWF, on the expression and purification of Green Fluorescence Protein (GFP) used as a model fusion protein. The titers obtained with the expression of these fusion proteins in soluble form were 0.11mgml -1 and 0.48mgml -1 for WFWFWF and NWNWNW, respectively. A combinatorial library comprising 64 ligands based on the Ugi reaction was prepared and screened for binding GFP-tagged and non-tagged proteins. Complementary ligands A2C2 and A3C1 were selected for the effective capture of NWNWNW and WFWFWF tagged proteins, respectively, in soluble forms. These affinity pairs displayed 10 6 M -1 affinity constants and Qmax values of 19.11±2.60ugg -1 and 79.39ugg -1 for the systems WFWFWF AND NWNWNW, respectively. GFP fused to the WFWFWF affinity tag was also produced as inclusion bodies, and a refolding-on column strategy was explored using the ligand A4C8, selected from the combinatorial library of ligands but in presence of denaturant agents. Copyright © 2016 Elsevier B.V. All rights reserved.

Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression

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Perera Rajika L

2004-12-01

Full Text Available Abstract Background In the search for generic expression strategies for mammalian protein families several bacterial expression vectors were examined for their ability to promote high yields of soluble protein. Proteins studied included cell surface receptors (Ephrins and Eph receptors, CD44, kinases (EGFR-cytoplasmic domain, CDK2 and 4, proteases (MMP1, CASP2, signal transduction proteins (GRB2, RAF1, HRAS and transcription factors (GATA2, Fli1, Trp53, Mdm2, JUN, FOS, MAD, MAX. Over 400 experiments were performed where expression of 30 full-length proteins and protein domains were evaluated with 6 different N-terminal and 8 C-terminal fusion partners. Expression of an additional set of 95 mammalian proteins was also performed to test the conclusions of this study. Results Several protein features correlated with soluble protein expression yield including molecular weight and the number of contiguous hydrophobic residues and low complexity regions. There was no relationship between successful expression and protein pI, grand average of hydropathicity (GRAVY, or sub-cellular location. Only small globular cytoplasmic proteins with an average molecular weight of 23 kDa did not require a solubility enhancing tag for high level soluble expression. Thioredoxin (Trx and maltose binding protein (MBP were the best N-terminal protein fusions to promote soluble expression, but MBP was most effective as a C-terminal fusion. 63 of 95 mammalian proteins expressed at soluble levels of greater than 1 mg/l as N-terminal H10-MBP fusions and those that failed possessed, on average, a higher molecular weight and greater number of contiguous hydrophobic amino acids and low complexity regions. Conclusions By analysis of the protein features identified here, this study will help predict which mammalian proteins and domains can be successfully expressed in E. coli as soluble product and also which are best targeted for a eukaryotic expression system. In some cases

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

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.

Improving recombinant protein purification yield

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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...

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.

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.

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

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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.

Protein solubility and folding enhancement by interaction with RNA.

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Seong Il Choi

Full Text Available While basic mechanisms of several major molecular chaperones are well understood, this machinery has been known to be involved in folding of only limited number of proteins inside the cells. Here, we report a chaperone type of protein folding facilitated by interaction with RNA. When an RNA-binding module is placed at the N-terminus of aggregation-prone target proteins, this module, upon binding with RNA, further promotes the solubility of passenger proteins, potentially leading to enhancement of proper protein folding. Studies on in vitro refolding in the presence of RNA, coexpression of RNA molecules in vivo and the mutants with impaired RNA binding ability suggests that RNA can exert chaperoning effect on their bound proteins. The results suggest that RNA binding could affect the overall kinetic network of protein folding pathway in favor of productive folding over off-pathway aggregation. In addition, the RNA binding-mediated solubility enhancement is extremely robust for increasing soluble yield of passenger proteins and could be usefully implemented for high-throughput protein expression for functional and structural genomic research initiatives. The RNA-mediated chaperone type presented here would give new insights into de novo folding in vivo.

Thermal precipitation fluorescence assay for protein stability screening.

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Fan, Junping; Huang, Bo; Wang, Xianping; Zhang, Xuejun C

2011-09-01

A simple and reliable method of protein stability assessment is desirable for high throughput expression screening of recombinant proteins. Here we described an assay termed thermal precipitation fluorescence (TPF) which can be used to compare thermal stabilities of recombinant protein samples directly from cell lysate supernatants. In this assay, target membrane proteins are expressed as recombinant fusions with a green fluorescence protein tag and solubilized with detergent, and the fluorescence signals are used to report the quantity of the fusion proteins in the soluble fraction of the cell lysate. After applying a heat shock, insoluble protein aggregates are removed by centrifugation. Subsequently, the amount of remaining protein in the supernatant is quantified by in-gel fluorescence analysis and compared to samples without a heat shock treatment. Over 60 recombinant membrane proteins from Escherichia coli were subject to this screening in the presence and absence of a few commonly used detergents, and the results were analyzed. Because no sophisticated protein purification is required, this TPF technique is suitable to high throughput expression screening of recombinant membrane proteins as well as soluble ones and can be used to prioritize target proteins based on their thermal stabilities for subsequent large scale expression and structural studies. Copyright © 2011 Elsevier Inc. All rights reserved.

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.

Gene Delivery into Plant Cells for Recombinant Protein Production

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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.

Recombinant DNA production of spider silk proteins.

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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.

Optimization of translation profiles enhances protein expression and solubility.

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Anne-Katrin Hess

Full Text Available mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5'-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein.

Yeast synthetic biology for the production of recombinant therapeutic proteins.

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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.

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.

Yeast expressed recombinant Hemagglutinin protein of Novel H1N1 elicits neutralising antibodies in rabbits and mice

Directory of Open Access Journals (Sweden)

Athmaram TN

2011-11-01

Full Text Available Abstract Currently available vaccines for the pandemic Influenza A (H1N1 2009 produced in chicken eggs have serious impediments viz limited availability, risk of allergic reactions and the possible selection of sub-populations differing from the naturally occurring virus, whereas the cell culture derived vaccines are time consuming and may not meet the demands of rapid global vaccination required to combat the present/future pandemic. Hemagglutinin (HA based subunit vaccine for H1N1 requires the HA protein in glycosylated form, which is impossible with the commonly used bacterial expression platform. Additionally, bacterial derived protein requires extensive purification and refolding steps for vaccine applications. For these reasons an alternative heterologous system for rapid, easy and economical production of Hemagglutinin protein in its glycosylated form is required. The HA gene of novel H1N1 A/California/04/2009 was engineered for expression in Pichia pastoris as a soluble secreted protein. The full length HA- synthetic gene having α-secretory tag was integrated into P. pastoris genome through homologous recombination. The resultant Pichia clones having multiple copy integrants of the transgene expressed full length HA protein in the culture supernatant. The Recombinant yeast derived H1N1 HA protein elicited neutralising antibodies both in mice and rabbits. The sera from immunised animals also exhibited Hemagglutination Inhibition (HI activity. Considering the safety, reliability and also economic potential of Pichia expression platform, our preliminary data indicates the feasibility of using this system as an alternative for large-scale production of recombinant influenza HA protein in the face of influenza pandemic threat.

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.

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

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....

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

Production of Active Nonglycosylated Recombinant B-Chain of Type-2 Ribosome-Inactivating Protein from Viscum articulatum and Its Biological Effects on Peripheral Blood Mononuclear Cells

Directory of Open Access Journals (Sweden)

Tzu-Li Lu

2011-01-01

Full Text Available Type-2 ribosome-inactivating proteins, composed of a toxic A-chain and lectin-like B-chain, display various biological functions, including cytotoxicity and immunomodulation. We here cloned the lectin-like B-chain encoding fragment of a newly identified type-2 RIP gene, articulatin gene, from Viscum articulatum, into a bacterial expression vector to obtain nonglycosylated recombinant protein expressed in inclusion bodies. After purification and protein refolding, soluble refolded recombinant articulatin B-chain (rATB showed lectin activity specific toward galactoside moiety and was stably maintained while stored in low ionic strength solution. Despite lacking glycosylation, rATB actively bound leukocytes with preferential binding to monocytes and in vitro stimulated PBMCs to release cytokines without obvious cytotoxicity. These results implicated such a B-chain fragment as a potential immunomodulator.

Protein Solubility as Quality Index for Processed Soybean

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Rodica Căpriţă

2010-05-01

Full Text Available Protein quality of soybean meal (SBM is linked to both the reduction of antinutritional factors (ANFs, and the optimization of protein digestibility. Both insufficient- and over-heating result in poor quality SBM. Inadequate heating fails to completely destroy the ANFs, which may have a detrimental impact on animal performance, while excessive heating reduces the availability of lysine via the Maillard reaction and possibly, to a lesser extent, of other amino acids. The objective of our study was to compare some biochemical laboratory procedures for assessing quality of SBM: urease index (UI, protein dispersibility index (PDI, KOH protein solubility (PS, and nitrogen solubility index (NSI. The experimental data reveal that UI is not useful to determine excessive heat treatment since additional heating has no effect on the urease index. KOH protein solubility remains high, during initial heat treatment. In marked contrast, the PDI and NSI decreased incrementally from 78% to 20% and from 97% to 60%, respectively, when heating 0 to 30 minutes. Combing the PDI test with the urease test could be useful to monitor soybean quality. SBM containing low UI (0.3 or below and high PDI (40 to 45% may indicate that the sample is definitely high quality because it has been adequately heat processed, but not overprocessed.

A Soluble, Folded Protein without Charged Amino Acid Residues

DEFF Research Database (Denmark)

Højgaard, Casper; Kofoed, Christian; Espersen, Roall

2016-01-01

side chains can maintain solubility, stability, and function. As a model, we used a cellulose-binding domain from Cellulomonas fimi, which, among proteins of more than 100 amino acids, presently is the least charged in the Protein Data Bank, with a total of only four titratable residues. We find......Charges are considered an integral part of protein structure and function, enhancing solubility and providing specificity in molecular interactions. We wished to investigate whether charged amino acids are indeed required for protein biogenesis and whether a protein completely free of titratable...... that the protein shows a surprising resilience toward extremes of pH, demonstrating stability and function (cellulose binding) in the pH range from 2 to 11. To ask whether the four charged residues present were required for these properties of this protein, we altered them to nontitratable ones. Remarkably...

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.

Dissection of an old protein reveals a novel application: domain D of Staphylococcus aureus Protein A (sSpAD as a secretion - tag

Directory of Open Access Journals (Sweden)

Paal Michael

2010-11-01

Full Text Available Abstract Background Escherichia coli as a frequently utilized host organism for recombinant protein production offers different cellular locations with distinct qualities. The periplasmic space is often favored for the production of complex proteins due to enhanced disulfide bond formation, increased target product stability and simplified downstream processing. To direct proteins to the periplasmic space rather small proteinaceus tags that can be used for affinity purification would be advantageous. Results We discovered that domain D of the Staphylococcus aureus protein A was sufficient for the secretion of various target proteins into the periplasmic space of E. coli. Our experiments indicated the Sec pathway as the mode of secretion, although N-terminal processing was not observed. Furthermore, the solubility of recombinant fusion proteins was improved for proteins prone to aggregation. The tag allowed a straightforward affinity purification of recombinant fusion protein via an IgG column, which was exemplified for the target protein human superoxide dismutase 1 (SOD. Conclusions In this work we present a new secretion tag that combines several advantages for the production of recombinant proteins in E. coli. Domain D of S. aureus protein A protects the protein of interest against N-terminal degradation, increases target protein solubility and enables a straight-forward purification of the recombinant protein using of IgG columns.

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.

Recombinant allergen Lol p II: expression, purification and characterization.

Science.gov (United States)

Tamborini, E; Brandazza, A; De Lalla, C; Musco, G; Siccardi, A G; Arosio, P; Sidoli, A

1995-05-01

Pollen from perennial rye grass (Lolium perenne) is a major cause of type I allergies worldwide. It contains complex mixtures of proteins, among which Lol p II is a major allergen. Previously, we have reported the cloning and sequencing of Lol p II and its expression in fusion with the heavy chain of human ferritin as carrier polypeptide (Sidoli et al., 1993, J. biol. Chem. 268, 21819-21825). Here, we describe the expression, purification and characterization of a recombinant Lol p II overproduced as a non-fusion protein in the periplasm of E. coli. The recombinant allergen was expressed in high yields and was easily purified in milligram amounts. It competed with the natural Lol p II for binding to specific IgE, and it induced allergic responses in skin prick tests, indicating to be immunologically analogous to the natural protein. Biochemical analyses indicate that recombinant Lol p II is a highly stable and soluble monomeric molecule which behaves like a small globular protein.

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.

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)

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

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.

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)

Limitations of polyethylene glycol-induced precipitation as predictive tool for protein solubility during formulation development.

Science.gov (United States)

Hofmann, Melanie; Winzer, Matthias; Weber, Christian; Gieseler, Henning

2018-05-01

Polyethylene glycol (PEG)-induced protein precipitation is often used to extrapolate apparent protein solubility at specific formulation compositions. The procedure was used for several fields of application such as protein crystal growth but also protein formulation development. Nevertheless, most studies focused on applicability in protein crystal growth. In contrast, this study focuses on applicability of PEG-induced precipitation during high-concentration protein formulation development. In this study, solubility of three different model proteins was investigated over a broad range of pH. Solubility values predicted by PEG-induced precipitation were compared to real solubility behaviour determined by either turbidity or content measurements. Predicted solubility by PEG-induced precipitation was confirmed for an Fc fusion protein and a monoclonal antibody. In contrast, PEG-induced precipitation failed to predict solubility of a single-domain antibody construct. Applicability of PEG-induced precipitation as indicator of protein solubility during formulation development was found to be not valid for one of three model molecules. Under certain conditions, PEG-induced protein precipitation is not valid for prediction of real protein solubility behaviour. The procedure should be used carefully as tool for formulation development, and the results obtained should be validated by additional investigations. © 2017 Royal Pharmaceutical Society.

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.)

The Influence of Tissue Lyophilization and Gamma Irradiation on the Solubility of Proteins

International Nuclear Information System (INIS)

Komender, J.; Jendyk, J.; Leibschang, J.

1967-01-01

Most recent methods of tissue preservation are based on lyophilization and sterilization with gamma rays. Unfortunately the tissues preserved by this method lose their viability, this being connected with protein denaturation. The denaturing influence either of lyophilization or sterilization with gamma rays on different materials has been described. However, no observations on denaturation of proteins in prepared grafts are known. The work aimed at establishing the influence of individual stages of the procedure used in a tissue bank on the solubility of proteins. An experiment was performed using rat liver as a model tissue. Solubility of protein was determined in five groups of material, as follows: (1) fresh tissue, used as a control, (2) frozen tissue, (3) frozen and lyophilized tissue, (4) frozen, lyophilized and irradiated tissue, and (5) fresh irradiated tissue. Folin's method was used for determination of protein in water extracts of tissues. It was found that: (1) the whole procedure considerably diminished the protein solubility, (2) freezing diminishes the protein solubility by 35% on average, (3) lyophilization causes no further protein denaturation, (4) protein solubility is reduced most (by about 65%) by sterilization with gamma rays. (author)

Optimization of the purification methods for recovery of recombinant growth hormone from Paralichthys olivaceus

Science.gov (United States)

Zang, Xiaonan; Zhang, Xuecheng; Mu, Xiaosheng; Liu, Bin

2013-03-01

This study aimed to optimize the purification of recombinant growth hormone from Paralichthys olivaceus. Recombinant flounder growth hormone (r-fGH) was expressed by Escherichia coli in form of inclusion body or as soluble protein under different inducing conditions. The inclusion body was renatured using two recovery methods, i.e., dilution and dialysis. Thereafter, the refolded protein was purified by Glutathione Sepharase 4B affinity chromatography and r-fGH was obtained by cleavage of thrombin. For soluble products, r-fGH was directly purified from the lysates by Glutathione Sepharase 4B affinity chromatography. ELISA-receptor assay demonstrated that despite its low receptor binding activity, the r-fGH purified from refolded inclusion body had a higher yield (2.605 mg L-1) than that from soluble protein (1.964 mg L-1). Of the tested recovery methods, addition of renaturing buffer (pH 8.5) into denatured inclusion body yielded the best recovery rate (17.9%). This work provided an optimized purification method for high recovery of r-fGH, thus contributing to the application of r-fGH to aquaculture.

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 "...

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

Optimization of Soluble Expression and Purification of Recombinant Human Rhinovirus Type-14 3C Protease Using Statistically Designed Experiments: Isolation and Characterization of the Enzyme.

Science.gov (United States)

Antoniou, Georgia; Papakyriacou, Irineos; Papaneophytou, Christos

2017-10-01

Human rhinovirus (HRV) 3C protease is widely used in recombinant protein production for various applications such as biochemical characterization and structural biology projects to separate recombinant fusion proteins from their affinity tags in order to prevent interference between these tags and the target proteins. Herein, we report the optimization of expression and purification conditions of glutathione S-transferase (GST)-tagged HRV 3C protease by statistically designed experiments. Soluble expression of GST-HRV 3C protease was initially optimized by response surface methodology (RSM), and a 5.5-fold increase in enzyme yield was achieved. Subsequently, we developed a new incomplete factorial (IF) design that examines four variables (bacterial strain, expression temperature, induction time, and inducer concentration) in a single experiment. The new design called Incomplete Factorial-Strain/Temperature/Time/Inducer (IF-STTI) was validated using three GST-tagged proteins. In all cases, IF-STTI resulted in only 10% lower expression yields than those obtained by RSM. Purification of GST-HRV 3C was optimized by an IF design that examines simultaneously the effect of the amount of resin, incubation time of cell lysate with resin, and glycerol and DTT concentration in buffers, and a further 15% increase in protease recovery was achieved. Purified GST-HRV 3C protease was active at both 4 and 25 °C in a variety of buffers.

Production of a Recombinant Dengue Virus 2 NS5 Protein and Potential Use as a Vaccine Antigen.

Science.gov (United States)

Alves, Rúbens Prince Dos Santos; Pereira, Lennon Ramos; Fabris, Denicar Lina Nascimento; Salvador, Felipe Scassi; Santos, Robert Andreata; Zanotto, Paolo Marinho de Andrade; Romano, Camila Malta; Amorim, Jaime Henrique; Ferreira, Luís Carlos de Souza

2016-06-01

Dengue fever is caused by any of the four known dengue virus serotypes (DENV1 to DENV4) that affect millions of people worldwide, causing a significant number of deaths. There are vaccines based on chimeric viruses, but they still are not in clinical use. Anti-DENV vaccine strategies based on nonstructural proteins are promising alternatives to those based on whole virus or structural proteins. The DENV nonstructural protein 5 (NS5) is the main target of anti-DENV T cell-based immune responses in humans. In this study, we purified a soluble recombinant form of DENV2 NS5 expressed in Escherichia coli at large amounts and high purity after optimization of expression conditions and purification steps. The purified DENV2 NS5 was recognized by serum from DENV1-, DENV2-, DENV3-, or DENV4-infected patients in an epitope-conformation-dependent manner. In addition, immunization of BALB/c mice with NS5 induced high levels of NS5-specific antibodies and expansion of gamma interferon- and tumor necrosis factor alpha-producing T cells. Moreover, mice immunized with purified NS5 were partially protected from lethal challenges with the DENV2 NGC strain and with a clinical isolate (JHA1). These results indicate that the recombinant NS5 protein preserves immunological determinants of the native protein and is a promising vaccine antigen capable of inducing protective immune responses. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Water-Soluble Chlorophyll Protein (WSCP) Stably Binds Two or Four Chlorophylls.

Science.gov (United States)

Palm, Daniel M; Agostini, Alessandro; Tenzer, Stefan; Gloeckle, Barbara M; Werwie, Mara; Carbonera, Donatella; Paulsen, Harald

2017-03-28

Water-soluble chlorophyll proteins (WSCPs) of class IIa from Brassicaceae form tetrameric complexes containing one chlorophyll (Chl) per apoprotein but no carotenoids. The complexes are remarkably stable toward dissociation and protein denaturation even at 100 °C and extreme pH values, and the Chls are partially protected against photooxidation. There are several hypotheses that explain the biological role of WSCPs, one of them proposing that they function as a scavenger of Chls set free upon plant senescence or pathogen attack. The biochemical properties of WSCP described in this paper are consistent with the protein acting as an efficient and flexible Chl scavenger. At limiting Chl concentrations, the recombinant WSCP apoprotein binds substoichiometric amounts of Chl (two Chls per tetramer) to form complexes that are as stable toward thermal dissociation, denaturation, and photodamage as the fully pigmented ones. If more Chl is added, these two-Chl complexes can bind another two Chls to reach the fully pigmented state. The protection of WSCP Chls against photodamage has been attributed to the apoprotein serving as a diffusion barrier for oxygen, preventing its access to triplet excited Chls and, thus, the formation of singlet oxygen. By contrast, the sequential binding of Chls by WSCP suggests a partially open or at least flexible structure, raising the question of how WSCP photoprotects its Chls without the help of carotenoids.

Evaluation of two recombinant Leishmania proteins identified by an immunoproteomic approach as tools for the serodiagnosis of canine visceral and human tegumentary leishmaniasis.

Science.gov (United States)

Coelho, Eduardo Antonio Ferraz; Costa, Lourena Emanuele; Lage, Daniela Pagliara; Martins, Vívian Tamietti; Garde, Esther; de Jesus Pereira, Nathália Cristina; Lopes, Eliane Gonçalves Paiva; Borges, Luiz Felipe Nunes Menezes; Duarte, Mariana Costa; Menezes-Souza, Daniel; de Magalhães-Soares, Danielle Ferreira; Chávez-Fumagalli, Miguel Angel; Soto, Manuel; Tavares, Carlos Alberto Pereira

2016-01-15

Serological diagnostic tests for canine and human leishmaniasis present problems related with their sensitivity and/or specificity. Recently, an immunoproteomic approach performed with Leishmania infantum proteins identified new parasite antigens. In the present study, the diagnostic properties of two of these proteins, cytochrome c oxidase and IgE-dependent histamine-releasing factor, were evaluated for the serodiagnosis of canine visceral (CVL) and human tegumentary (HTL) leishmaniasis. For the CVL diagnosis, sera samples from non-infected dogs living in an endemic or non-endemic area of leishmaniasis, sera from asymptomatic or symptomatic visceral leishmaniasis (VL) dogs, from Leish-Tec(®)-vaccinated dogs, and sera from animals experimentally infected by Trypanosoma cruzi or Ehrlichia canis were used. For the HTL diagnosis, sera from non-infected subjects living in an endemic area of leishmaniasis, sera from active cutaneous or mucosal leishmaniasis patients, as well as those from T. cruzi-infected patients were employed. ELISA assays using the recombinant proteins showed both sensitivity and specificity values of 100% for the serodiagnosis of both forms of disease, with high positive and negative predictive values, showing better diagnostic properties than the parasite recombinant A2 protein or a soluble Leishmania antigen extract. In this context, the two new recombinant proteins could be considered to be used in the serodiagnosis of CVL and HTL. Copyright © 2015 Elsevier B.V. All rights reserved.

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

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

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.

Enhanced production and purification of recombinant surface array protein (Sap) for use in detection of Bacillus anthracis.

Science.gov (United States)

Puranik, Nidhi; Tripathi, N K; Pal, V; Goel, Ajay Kumar

2018-05-01

Surface array protein (Sap) can be an important biomarker for specific detection of Bacillus anthracis , which is released by the bacterium during its growth in culture broth. In the present work, we have cloned and expressed Sap in Escherichia coli . The culture conditions and cultivation media were optimized and used in batch fermentation process for scale up of Sap in soluble form. The recombinant Sap was purified employing affinity chromatography followed by diafiltration. The final yield of purified protein was 20 and 46 mg/l of culture during shake flasks and batch fermentation, respectively. The protein purity and its reactivity were confirmed employing SDS-PAGE and Western blot, respectively. The antibodies raised against purified Sap were evaluated by Western blotting for detection of Sap released by B. anthracis . Our results showed that the Sap could be a novel marker for detection and confirmation of B. anthracis .

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).

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.

[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.

Recombinant IκBα-loaded curcumin nanoparticles for improved cancer therapeutics

International Nuclear Information System (INIS)

Banerjee, Subhamoy; Ghosh, Siddhartha Sankar; Sahoo, Amaresh Kumar; Chattopadhyay, Arun

2014-01-01

The field of recombinant protein therapeutics has been evolving rapidly, making significant impact on clinical applications for several diseases, including cancer. However, the functional aspects of proteins rely exclusively on their structural integrity, in which nanoparticle mediated delivery offers unique advantages over free proteins. In the present work, a novel strategy has been developed where the nanoparticles (NPs) used for the delivery of the recombinant protein could contribute to enhancing the therapeutic efficacy of the recombinant protein. The transcription factor, NFκB, involved in cell growth and its inhibitor, IκBα, regulates its proliferation. Another similar naturally available molecule, which inhibits the function of NFκB, is curcumin. Hence, we have developed a ‘green synthesis’ method for preparing water-soluble curcumin nanoparticles to stabilize recombinant IκBα protein. The NPs were characterized by UV–vis and fluorescence spectroscopy, transmission electron microscopy (TEM) and dynamic light scattering before administration into human cervical carcinoma (HeLa) and glioblastoma (U87MG) cells. Experimental results demonstrated that this combined module had enhanced therapeutic efficacy, causing apoptotic cell death, which was confirmed by cytotoxicity assay and flowcytometry analyses. The expression of apoptotic genes studied by semi-quantitative reverse transcription PCR delineated the molecular pathways involved in cell death. Thus, our study revealed that the functional delivery of recombinant IκBα-loaded curcumin NPs has promise as a natural-product-based protein therapeutics against cancer cells. (paper)

Recombinant IκBα-loaded curcumin nanoparticles for improved cancer therapeutics

Science.gov (United States)

Banerjee, Subhamoy; Sahoo, Amaresh Kumar; Chattopadhyay, Arun; Sankar Ghosh, Siddhartha

2014-08-01

The field of recombinant protein therapeutics has been evolving rapidly, making significant impact on clinical applications for several diseases, including cancer. However, the functional aspects of proteins rely exclusively on their structural integrity, in which nanoparticle mediated delivery offers unique advantages over free proteins. In the present work, a novel strategy has been developed where the nanoparticles (NPs) used for the delivery of the recombinant protein could contribute to enhancing the therapeutic efficacy of the recombinant protein. The transcription factor, NFκB, involved in cell growth and its inhibitor, IκBα, regulates its proliferation. Another similar naturally available molecule, which inhibits the function of NFκB, is curcumin. Hence, we have developed a ‘green synthesis’ method for preparing water-soluble curcumin nanoparticles to stabilize recombinant IκBα protein. The NPs were characterized by UV-vis and fluorescence spectroscopy, transmission electron microscopy (TEM) and dynamic light scattering before administration into human cervical carcinoma (HeLa) and glioblastoma (U87MG) cells. Experimental results demonstrated that this combined module had enhanced therapeutic efficacy, causing apoptotic cell death, which was confirmed by cytotoxicity assay and flowcytometry analyses. The expression of apoptotic genes studied by semi-quantitative reverse transcription PCR delineated the molecular pathways involved in cell death. Thus, our study revealed that the functional delivery of recombinant IκBα-loaded curcumin NPs has promise as a natural-product-based protein therapeutics against cancer cells.

Soluble Non-ammonia Nitrogen in Ruminal and Omasal Digesta of Korean Native Steers Supplemented with Soluble Proteins

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C. W. Choi

2012-09-01

Full Text Available An experiment was conducted to study the effect of soluble protein supplements on concentration of soluble non-ammonia nitrogen (SNAN in the liquid phase of ruminal (RD and omasal digesta (OD of Korean native steers, and to investigate diurnal pattern in SNAN concentration in RD and OD. Three ruminally cannulated Korean native steers in a 3×3 Latin square design consumed a basal diet of rice straw and corn-based concentrate (control, and that supplemented (kg/d DM basis with intact casein (0.24; IC or acid hydrolyzed casein (0.46; AHC. Ruminal digesta was sampled using a vacuum pump, whereas OD was collected using an omasal sampling system at 2.0 h intervals after a morning feeding. The SNAN fractions (free amino acid (AA, peptide and soluble protein in RD and OD were assessed using the ninhydrin assay. Concentrations of free AA and total SNAN in RD were significantly (p<0.05 lower than those in OD. Although free AA concentration was relatively high, mean peptide was quantitatively the most important fraction of total SNAN in both RD and OD, indicating that degradation of peptide to AA rather than hydrolysis of soluble protein to peptide or deamination may be the most limiting step in rumen proteolysis of Korean native steers. Diurnal variation in peptide concentration in OD for the soluble protein supplemented diets during the feeding cycle peaked 2 h post-feeding and decreased thereafter whereas that for the control was relatively constant during the entire feeding cycle. Diurnal variation in peptide concentration was rather similar between RD and OD.

Combination therapy with sivelestat and recombinant human soluble thrombomodulin for ARDS and DIC patients

Directory of Open Access Journals (Sweden)

Miyoshi S

2014-09-01

Full Text Available Seigo Miyoshi,1 Ryoji Ito,1 Hitoshi Katayama,1 Kentaro Dote,2 Mayuki Aibiki,3 Hironobu Hamada,1,4 Takafumi Okura,1 Jitsuo Higaki1 1Department of Cardiology, Pulmonology, Hypertension and Nephrology, Ehime University Graduate School of Medicine, 2Intensive Care Division, Ehime University Hospital, 3Department of Emergency and Critical Care Medicine, School of Medicine, Ehime University, Shitsukawa, Toon, Ehime, 4Department of Physical Analysis and Therapeutic Sciences, Graduate School of Biomedical and Health Sciences, Hiroshima University, Kasumi, Minami-ku, Hiroshima, Japan Background: Neutrophil elastase, alveolar thrombin generation, and fibrin deposition play crucial roles in the development of acute respiratory distress syndrome (ARDS and disseminated intravascular coagulation (DIC. However, the usefulness of combination therapy with a selective neutrophil elastase inhibitor, sivelestat, and recombinant human soluble thrombomodulin (rhTM for patients with ARDS and DIC remains unknown. Methods: We conducted a retrospective data analysis of 142 ARDS patients with DIC to assess the effects of sivelestat combined with rhTM. Patients were divided into four groups: control (no sivelestat or rhTM treatment, sivelestat treatment alone, rhTM treatment alone, and combined treatment with sivelestat and rhTM. A Cox proportional hazard model was used to assess subject mortality rates. The efficacy of these drugs was evaluated based on survival rate, number of ventilator-free days, and change in PaO2/FIO2 (P/F ratios and DIC scores before and at 7 days after a diagnosis of ARDS with DIC. Results: Multivariate analysis showed that patient age, combination therapy, gas exchange, organ failure, cause, associated disease score, and serum C-reactive protein levels were predictors of mortality for patients with ARDS and DIC. As compared with untreated controls, combination therapy significantly improved the 60-day survival rate of patients with ARDS and DIC

Cloning of human tumor necrosis factor (TNF) receptor cDNA and expression of recombinant soluble TNF-binding protein

International Nuclear Information System (INIS)

Gray, P.W.; Barrett, K.; Chantry, D.; Turner, M.; Feldmann, M.

1990-01-01

The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extracellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10 -9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ)

Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

Science.gov (United States)

Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

1990-10-01

The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extra-cellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ).

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

Science.gov (United States)

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

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

Cytokine-like factor-1, a novel soluble protein, shares homology with members of the cytokine type I receptor family.

Science.gov (United States)

Elson, G C; Graber, P; Losberger, C; Herren, S; Gretener, D; Menoud, L N; Wells, T N; Kosco-Vilbois, M H; Gauchat, J F

1998-08-01

In this report we describe the identification, cloning, and expression pattern of human cytokine-like factor 1 (hCLF-1) and the identification and cloning of its murine homologue. They were identified from expressed sequence tags using amino acid sequences from conserved regions of the cytokine type I receptor family. Human CLF-1 and murine CLF-1 shared 96% amino acid identity and significant homology with many cytokine type I receptors. CLF-1 is a secreted protein, suggesting that it is either a soluble subunit within a cytokine receptor complex, like the soluble form of the IL-6R alpha-chain, or a subunit of a multimeric cytokine, e.g., IL-12 p40. The highest levels of hCLF-1 mRNA were observed in lymph node, spleen, thymus, appendix, placenta, stomach, bone marrow, and fetal lung, with constitutive expression of CLF-1 mRNA detected in a human kidney fibroblastic cell line. In fibroblast primary cell cultures, CLF-1 mRNA was up-regulated by TNF-alpha, IL-6, and IFN-gamma. Western blot analysis of recombinant forms of hCLF-1 showed that the protein has the tendency to form covalently linked di- and tetramers. These results suggest that CLF-1 is a novel soluble cytokine receptor subunit or part of a novel cytokine complex, possibly playing a regulatory role in the immune system and during fetal development.

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.

In vivo modification of tyrosine residues in recombinant mussel adhesive protein by tyrosinase co-expression in Escherichia coli

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Choi Yoo

2012-10-01

Full Text Available Abstract Background In nature, mussel adhesive proteins (MAPs show remarkable adhesive properties, biocompatibility, and biodegradability. Thus, they have been considered promising adhesive biomaterials for various biomedical and industrial applications. However, limited production of natural MAPs has hampered their practical applications. Recombinant production in bacterial cells could be one alternative to obtain useable amounts of MAPs, although additional post-translational modification of tyrosine residues into 3,4-dihydroxyphenyl-alanine (Dopa and Dopaquinone is required. The superior properties of MAPs are mainly attributed to the introduction of quinone-derived intermolecular cross-links. To solve this problem, we utilized a co-expression strategy of recombinant MAP and tyrosinase in Escherichia coli to successfully modify tyrosine residues in vivo. Results A recombinant hybrid MAP, fp-151, was used as a target for in vivo modification, and a dual vector system of pET and pACYC-Duet provided co-expression of fp-151 and tyrosinase. As a result, fp-151 was over-expressed and mainly obtained from the soluble fraction in the co-expression system. Without tyrosinase co-expression, fp-151 was over-expressed in an insoluble form in inclusion bodies. The modification of tyrosine residues in the soluble-expressed fp-151 was clearly observed from nitroblue tetrazolium staining and liquid-chromatography-mass/mass spectrometry analyses. The purified, in vivo modified, fp-151 from the co-expression system showed approximately 4-fold higher bulk-scale adhesive strength compared to in vitro tyrosinase-treated fp-151. Conclusion Here, we reported a co-expression system to obtain in vivo modified MAP; additional in vitro tyrosinase modification was not needed to obtain adhesive properties and the in vivo modified MAP showed superior adhesive strength compared to in vitro modified protein. It is expected that this co-expression strategy will accelerate

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....

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.

Affinity-based, biophysical methods to detect and analyze ligand binding to recombinant proteins: matching high information content with high throughput.

Science.gov (United States)

Holdgate, Geoff A; Anderson, Malcolm; Edfeldt, Fredrik; Geschwindner, Stefan

2010-10-01

Affinity-based technologies have become impactful tools to detect, monitor and characterize molecular interactions using recombinant target proteins. This can aid the understanding of biological function by revealing mechanistic details, and even more importantly, enables the identification of new improved ligands that can modulate the biological activity of those targets in a desired fashion. The selection of the appropriate technology is a key step in that process, as each one of the currently available technologies offers a characteristic type of biophysical information about the ligand-binding event. Alongside the indisputable advantages of each of those technologies they naturally display diverse restrictions that are quite frequently related to the target system to be studied but also to the affinity, solubility and molecular size of the ligands. This paper discusses some of the theoretical and experimental aspects of the most common affinity-based methods, what type of information can be gained from each one of those approaches, and what requirements as well as limitations are expected from working with recombinant proteins on those platforms and how those can be optimally addressed.

Novel mitochondria-targeted heat-soluble proteins identified in the anhydrobiotic Tardigrade improve osmotic tolerance of human cells.

Directory of Open Access Journals (Sweden)

Sae Tanaka

Full Text Available Tardigrades are able to tolerate almost complete dehydration through transition to a metabolically inactive state, called "anhydrobiosis". Late Embryogenesis Abundant (LEA proteins are heat-soluble proteins involved in the desiccation tolerance of many anhydrobiotic organisms. Tardigrades, Ramazzottius varieornatus, however, express predominantly tardigrade-unique heat-soluble proteins: CAHS (Cytoplasmic Abundant Heat Soluble and SAHS (Secretory Abundant Heat Soluble proteins, which are secreted or localized in most intracellular compartments, except the mitochondria. Although mitochondrial integrity is crucial to ensure cellular survival, protective molecules for mitochondria have remained elusive. Here, we identified two novel mitochondrial heat-soluble proteins, RvLEAM and MAHS (Mitochondrial Abundant Heat Soluble, as potent mitochondrial protectants from Ramazzottius varieornatus. RvLEAM is a group3 LEA protein and immunohistochemistry confirmed its mitochondrial localization in tardigrade cells. MAHS-green fluorescent protein fusion protein localized in human mitochondria and was heat-soluble in vitro, though no sequence similarity with other known proteins was found, and one region was conserved among tardigrades. Furthermore, we demonstrated that RvLEAM protein as well as MAHS protein improved the hyperosmotic tolerance of human cells. The findings of the present study revealed that tardigrade mitochondria contain at least two types of heat-soluble proteins that might have protective roles in water-deficient environments.

Profiling and relationship of water-soluble sugar and protein compositions in soybean seeds.

Science.gov (United States)

Yu, Xiaomin; Yuan, Fengjie; Fu, Xujun; Zhu, Danhua

2016-04-01

Sugar and protein are important quality traits in soybean seeds for making soy-based food products. However, the investigations on both compositions and their relationship have rarely been reported. In this study, a total of 35 soybean germplasms collected from Zhejiang province of China, were evaluated for both water-soluble sugar and protein. The total water-soluble sugar (TWSS) content of the germplasms studied ranged from 84.70 to 140.91 mg/g and the water-soluble protein (WSP) content varied from 26.5% to 36.0%. The WSP content showed positive correlations with the TWSS and sucrose contents but negative correlations with the fructose and glucose contents. The clustering showed the 35 germplasms could be divided into four groups with specific contents of sugar and protein. The combination of water-soluble sugar and protein profiles provides useful information for future breeding and genetic research. This investigation will facilitate future work for seed quality improvement. Copyright © 2015. Published by Elsevier Ltd.

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.

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

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

DIFFERENTIAL BINDING OF HUMAN INTERLEUKIN-1 (IL-1) RECEPTOR ANTAGONIST TO NATURAL AND RECOMBINANT SOLUBLE AND CELLULAR IL-1 TYPE-I RECEPTORS

DEFF Research Database (Denmark)

Svenson, Morten; Nedergaard, Susanne; Heegaard, Peter M. H.

1995-01-01

antagonist (IL-1ra). Recombinant soluble human IL-1RI expressed in COS cells (sIL-1RI) consists of the extracellular part of the receptor and binds all three known IL-1 species but preferentially to IL-1ra. We further characterized the sizes and binding of IL-1raBF and sIL-1RI to IL-1ra by polyacrylamide gel...... electrophoresis in the presence of sodium dodecylsulfate, ligand binding interference analyses, N-glycosidase treatment, concanavalin A affinity chromatography, and with the use of monoclonal antibodies (mAb) to human recombinant IL-1ra. We also evaluated the binding of IL-1ra to cellular IL-1RI on MRC5...... binding of both molecules to IL-1ra. Both factors blocked binding of IL-1ra to cellular IL-1RI, as did mAb to IL-1ra, but the sites on IL-1ra which bound to the mAb, and to IL-1raBF and sIL-1RI, differed. We conclude that there are important differences between the natural and recombinant forms of soluble...

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.

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; ...

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

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.

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.

Soluble protein isolated from low cost fish and fish wastes

OpenAIRE

Lekshmy Nair, A.; Gopakumar, K.

1982-01-01

The method of preparation, composition, amino acid content, protein efficiency ratio and areas of possible application of water soluble protein isolates from low cost fish and fish wastes are discussed in detail in this communication.

Effects of gamma irradiation on physicochemical properties of heat-induced gel prepared with chicken salt-soluble proteins

International Nuclear Information System (INIS)

Choi, Yun-Sang; Kim, Hyun-Wook; Hwang, Ko-Eun; Song, Dong-Heon; Jeong, Tae-Jun; Seo, Kwang-Wook; Kim, Young-Boong; Kim, Cheon-Jei

2015-01-01

The technological effects of gamma irradiation (0, 3, 7, and 10 kGy) on chicken salt-soluble meat proteins in a model system were investigated. There were no significant differences in protein, fat, and ash content, and sarcoplasmic protein solubility among all samples. The samples with increasing gamma irradiation levels had higher pH, lightness, yellowness, and apparent viscosity, whereas moisture content, water holding capacity, redness, myofibrillar protein solubility, total protein solubility, hardness, springiness, cohesiveness, gumminess, and chewiness were the highest in the unirradiated control. The result from meat products using gamma irradiation was intended to provide a basic resource processing technology. - Highlights: • The effect of gamma irradiation on salt-soluble meat proteins was investigated. • Gelling properties of salt-soluble protein affected by gamma irradiation. • Gamma irradiation of meat products provides a basic resource processing technology

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

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

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....

Changes in protein solubility, fermentative capacity, viscoelasticity ...

African Journals Online (AJOL)

Frozen dough should be stored for fewer than 21 days; time in which the loaf volume of bread made from frozen dough was approximately 40.84% smaller than that of fresh bread dough formulation. Keywords: French type bread, frozen dough, protein solubility, baking quality, viscoelasticity. African Journal of Biotechnology ...

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.

Heterologous Expression of the Carrot Hsp17.7 gene Increased Growth, Cell Viability, and Protein Solubility in Transformed Yeast (Saccharomyces cerevisiae) under Heat, Cold, Acid, and Osmotic Stress Conditions.

Science.gov (United States)

Ko, Eunhye; Kim, Minhye; Park, Yunho; Ahn, Yeh-Jin

2017-08-01

In industrial fermentation of yeast (Saccharomyces cerevisiae), culture conditions are often modified from the optimal growth conditions of the cells to maintain large-scale cultures and/or to increase recombinant protein production. However, altered growth conditions can be stressful to yeast cells resulting in reduced cell growth and viability. In this study, a small heat shock protein gene from carrot (Daucus carota L.), Hsp17.7, was inserted into the yeast genome via homologous recombination to increase tolerance to stress conditions that can occur during industrial culture. A DNA construct, Translational elongation factor gene promoter-carrot Hsp17.7 gene-Phosphoribosyl-anthranilate isomerase gene (an auxotrophic marker), was generated by a series of PCRs and introduced into the chromosome IV of the yeast genome. Immunoblot analysis showed that carrot Hsp17.7 accumulated in the transformed yeast cell lines. Growth rates and cell viability of these cell lines were higher than control cell lines under heat, cold, acid, and hyperosmotic stress conditions. Soluble protein levels were higher in the transgenic cell lines than control cell lines under heat and cold conditions, suggesting the molecular chaperone function of the recombinant Hsp17.7. This study showed that a recombinant DNA construct containing a HSP gene from carrot was successfully expressed in yeast by homologous recombination and increased tolerances to abiotic stress conditions.

Colon-specific delivery of a probiotic-derived soluble protein ameliorates intestinal inflammation in mice through an EGFR-dependent mechanism

Science.gov (United States)

Yan, Fang; Cao, Hanwei; Cover, Timothy L.; Washington, M. Kay; Shi, Yan; Liu, LinShu; Chaturvedi, Rupesh; Peek, Richard M.; Wilson, Keith T.; Polk, D. Brent

2011-01-01

Probiotic bacteria can potentially have beneficial effects on the clinical course of several intestinal disorders, but our understanding of probiotic action is limited. We have identified a probiotic bacteria–derived soluble protein, p40, from Lactobacillus rhamnosus GG (LGG), which prevents cytokine-induced apoptosis in intestinal epithelial cells. In the current study, we analyzed the mechanisms by which p40 regulates cellular responses in intestinal epithelial cells and p40’s effects on experimental colitis using mouse models. We show that the recombinant p40 protein activated EGFR, leading to Akt activation. Activation of EGFR by p40 was required for inhibition of cytokine-induced apoptosis in intestinal epithelial cells in vitro and ex vivo. Furthermore, we developed a pectin/zein hydrogel bead system to specifically deliver p40 to the mouse colon, which activated EGFR in colon epithelial cells. Administration of p40-containing beads reduced intestinal epithelial apoptosis and disruption of barrier function in the colon epithelium in an EGFR-dependent manner, thereby preventing and treating DSS-induced intestinal injury and acute colitis. Furthermore, p40 activation of EGFR was required for ameliorating colon epithelial cell apoptosis and chronic inflammation in oxazolone-induced colitis. These data define what we believe to be a previously unrecognized mechanism of probiotic-derived soluble proteins in protecting the intestine from injury and inflammation. PMID:21606592

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.

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.

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.

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.

Expression and purification of short hydrophobic elastin-like polypeptides with maltose-binding protein as a solubility tag.

Science.gov (United States)

Bataille, Laure; Dieryck, Wilfrid; Hocquellet, Agnès; Cabanne, Charlotte; Bathany, Katell; Lecommandoux, Sébastien; Garbay, Bertrand; Garanger, Elisabeth

2015-06-01

Elastin-like polypeptides (ELPs) are biodegradable polymers with interesting physico-chemical properties for biomedical and biotechnological applications. The recombinant expression of hydrophobic elastin-like polypeptides is often difficult because they possess low transition temperatures, and therefore form aggregates at sub-ambient temperatures. To circumvent this difficulty, we expressed in Escherichia coli three hydrophobic ELPs (VPGIG)n with variable lengths (n=20, 40, and 60) in fusion with the maltose-binding protein (MBP). Fusion proteins were soluble and yields of purified MBP-ELP ranged between 66 and 127mg/L culture. After digestion of the fusion proteins by enterokinase, the ELP moiety was purified by using inverse transition cycling. The purified fraction containing ELP40 was slightly contaminated by traces of undigested fusion protein. Purification of ELP60 was impaired because of co-purification of the MBP tag during inverse transition cycling. ELP20 was successfully purified to homogeneity, as assessed by gel electrophoresis and mass spectrometry analyses. The transition temperature of ELP20 was measured at 15.4°C in low salt buffer. In conclusion, this method can be used to produce hydrophobic ELP of low molecular mass. Copyright © 2015 Elsevier Inc. All rights reserved.

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.

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

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.

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

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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.

Nuclear delivery of recombinant OCT4 by chitosan nanoparticles for transgene-free generation of protein-induced pluripotent stem cells.

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Tammam, Salma; Malak, Peter; Correa, Daphne; Rothfuss, Oliver; Azzazy, Hassan M E; Lamprecht, Alf; Schulze-Osthoff, Klaus

2016-06-21

Protein-based reprogramming of somatic cells is a non-genetic approach for the generation of induced pluripotent stem cells (iPSCs), whereby reprogramming factors, such as OCT4, SOX2, KLF4 and c-MYC, are delivered as functional proteins. The technique is considered safer than transgenic methods, but, unfortunately, most protein-based protocols provide very low reprogramming efficiencies. In this study, we developed exemplarily a nanoparticle (NP)-based delivery system for the reprogramming factor OCT4. To this end, we expressed human OCT4 in Sf9 insect cells using a baculoviral expression system. Recombinant OCT4 showed nuclear localization in Sf9 cells indicating proper protein folding. In comparison to soluble OCT4 protein, encapsulation of OCT4 in nuclear-targeted chitosan NPs strongly stabilized its DNA-binding activity even under cell culture conditions. OCT4-loaded NPs enabled cell treatment with high micromolar concentrations of OCT4 and successfully delivered active OCT4 into human fibroblasts. Chitosan NPs therefore provide a promising tool for the generation of transgene-free iPSCs.

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

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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.

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

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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.

Overview of the purification of recombinant proteins.

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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.

Recombinant expression and functional analysis of proteases from Streptococcus pneumoniae, Bacillus anthracis, and Yersinia pestis

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Pieper Rembert

2011-05-01

Full Text Available Abstract Background Uncharacterized proteases naturally expressed by bacterial pathogens represents important topic in infectious disease research, because these enzymes may have critical roles in pathogenicity and cell physiology. It has been observed that cloning, expression and purification of proteases often fail due to their catalytic functions which, in turn, cause toxicity in the E. coli heterologous host. Results In order to address this problem systematically, a modified pipeline of our high-throughput protein expression and purification platform was developed. This included the use of a specific E. coli strain, BL21(DE3 pLysS to tightly control the expression of recombinant proteins and various expression vectors encoding fusion proteins to enhance recombinant protein solubility. Proteases fused to large fusion protein domains, maltosebinding protein (MBP, SP-MBP which contains signal peptide at the N-terminus of MBP, disulfide oxidoreductase (DsbA and Glutathione S-transferase (GST improved expression and solubility of proteases. Overall, 86.1% of selected protease genes including hypothetical proteins were expressed and purified using a combination of five different expression vectors. To detect novel proteolytic activities, zymography and fluorescence-based assays were performed and the protease activities of more than 46% of purified proteases and 40% of hypothetical proteins that were predicted to be proteases were confirmed. Conclusions Multiple expression vectors, employing distinct fusion tags in a high throughput pipeline increased overall success rates in expression, solubility and purification of proteases. The combinatorial functional analysis of the purified proteases using fluorescence assays and zymography confirmed their function.

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

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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.

Simplified Method for Rapid Purification of Soluble Histones

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Nives Ivić

2016-06-01

Full Text Available Functional and structural studies of histone-chaperone complexes, nucleosome modifications, their interactions with remodelers and regulatory proteins rely on obtaining recombinant histones from bacteria. In the present study, we show that co-expression of Xenopus laevis histone pairs leads to production of soluble H2AH2B heterodimer and (H3H42 heterotetramer. The soluble histone complexes are purified by simple chromatographic techniques. Obtained H2AH2B dimer and H3H4 tetramer are proficient in histone chaperone binding and histone octamer and nucleosome formation. Our optimized protocol enables rapid purification of multiple soluble histone variants with a remarkable high yield and simplifies histone octamer preparation. We expect that this simple approach will contribute to the histone chaperone and chromatin research. This work is licensed under a Creative Commons Attribution 4.0 International License.

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 ...

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

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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.

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

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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.

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

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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.

Soluble Milk Protein Supplementation with Moderate Physical Activity Improves Locomotion Function in Aging Rats.

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Aude Lafoux

Full Text Available Aging is associated with a loss of muscle mass and functional capacity. Present study was designed to compare the impact of specific dairy proteins on muscular function with or without a low-intensity physical activity program on a treadmill in an aged rat model. We investigated the effects of nutritional supplementation, five days a week over a 2-month period with a slow digestible protein, casein or fast digestible proteins, whey or soluble milk protein, on strength and locomotor parameters in sedentary or active aged Wistar RjHan rats (17-19 months of age. An extensive gait analysis was performed before and after protein supplementation. After two months of protein administration and activity program, muscle force was evaluated using a grip test, spontaneous activity using an open-field and muscular mass by specific muscle sampling. When aged rats were supplemented with proteins without exercise, only minor effects of different diets on muscle mass and locomotion were observed: higher muscle mass in the casein group and improvement of stride frequencies with soluble milk protein. By contrast, supplementation with soluble milk protein just after physical activity was more effective at improving overall skeletal muscle function in old rats compared to casein. For active old rats supplemented with soluble milk protein, an increase in locomotor activity in the open field and an enhancement of static and dynamic gait parameters compared to active groups supplemented with casein or whey were observed without any differences in muscle mass and forelimb strength. These results suggest that consumption of soluble milk protein as a bolus immediately after a low intensity physical activity may be a suitable nutritional intervention to prevent decline in locomotion in aged rats and strengthen the interest to analyze the longitudinal aspect of locomotion in aged rodents.

Soluble Milk Protein Supplementation with Moderate Physical Activity Improves Locomotion Function in Aging Rats.

Science.gov (United States)

Lafoux, Aude; Baudry, Charlotte; Bonhomme, Cécile; Le Ruyet, Pascale; Huchet, Corinne

2016-01-01

Aging is associated with a loss of muscle mass and functional capacity. Present study was designed to compare the impact of specific dairy proteins on muscular function with or without a low-intensity physical activity program on a treadmill in an aged rat model. We investigated the effects of nutritional supplementation, five days a week over a 2-month period with a slow digestible protein, casein or fast digestible proteins, whey or soluble milk protein, on strength and locomotor parameters in sedentary or active aged Wistar RjHan rats (17-19 months of age). An extensive gait analysis was performed before and after protein supplementation. After two months of protein administration and activity program, muscle force was evaluated using a grip test, spontaneous activity using an open-field and muscular mass by specific muscle sampling. When aged rats were supplemented with proteins without exercise, only minor effects of different diets on muscle mass and locomotion were observed: higher muscle mass in the casein group and improvement of stride frequencies with soluble milk protein. By contrast, supplementation with soluble milk protein just after physical activity was more effective at improving overall skeletal muscle function in old rats compared to casein. For active old rats supplemented with soluble milk protein, an increase in locomotor activity in the open field and an enhancement of static and dynamic gait parameters compared to active groups supplemented with casein or whey were observed without any differences in muscle mass and forelimb strength. These results suggest that consumption of soluble milk protein as a bolus immediately after a low intensity physical activity may be a suitable nutritional intervention to prevent decline in locomotion in aged rats and strengthen the interest to analyze the longitudinal aspect of locomotion in aged rodents.

Galectin-1 as a fusion partner for the production of soluble and folded human {beta}-1,4-galactosyltransferase-T7 in E. coli

Energy Technology Data Exchange (ETDEWEB)

Pasek, Marta [Structural Glycobiology Section, SAIC-Frederick, Inc., Center for Cancer Research Nanobiology Program, Center for Cancer Research, NCI-Frederick, Frederick, MD 2170 (United States); Boeggeman, Elizabeth; Ramakrishnan, Boopathy [Structural Glycobiology Section, SAIC-Frederick, Inc., Center for Cancer Research Nanobiology Program, Center for Cancer Research, NCI-Frederick, Frederick, MD 2170 (United States); Basic Science Program, SAIC-Frederick, Inc., Center for Cancer Research Nanobiology Program, Center for Cancer Research, NCI-Frederick, Frederick, MD 2170 (United States); Qasba, Pradman K., E-mail: qasba@helix.nih.gov [Structural Glycobiology Section, SAIC-Frederick, Inc., Center for Cancer Research Nanobiology Program, Center for Cancer Research, NCI-Frederick, Frederick, MD 2170 (United States)

2010-04-09

The expression of recombinant proteins in Escherichia coli often leads to inactive aggregated proteins known as the inclusion bodies. To date, the best available tool has been the use of fusion tags, including the carbohydrate-binding protein; e.g., the maltose-binding protein (MBP) that enhances the solubility of recombinant proteins. However, none of these fusion tags work universally with every partner protein. We hypothesized that galectins, which are also carbohydrate-binding proteins, may help as fusion partners in folding the mammalian proteins in E. coli. Here we show for the first time that a small soluble lectin, human galectin-1, one member of a large galectin family, can function as a fusion partner to produce soluble folded recombinant human glycosyltransferase, {beta}-1,4-galactosyltransferase-7 ({beta}4Gal-T7), in E. coli. The enzyme {beta}4Gal-T7 transfers galactose to xylose during the synthesis of the tetrasaccharide linker sequence attached to a Ser residue of proteoglycans. Without a fusion partner, {beta}4Gal-T7 is expressed in E. coli as inclusion bodies. We have designed a new vector construct, pLgals1, from pET-23a that includes the sequence for human galectin-1, followed by the Tev protease cleavage site, a 6x His-coding sequence, and a multi-cloning site where a cloned gene is inserted. After lactose affinity column purification of galectin-1-{beta}4Gal-T7 fusion protein, the unique protease cleavage site allows the protein {beta}4Gal-T7 to be cleaved from galectin-1 that binds and elutes from UDP-agarose column. The eluted protein is enzymatically active, and shows CD spectra comparable to the folded {beta}4Gal-T1. The engineered galectin-1 vector could prove to be a valuable tool for expressing other proteins in E. coli.

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.

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.

Formation of soluble amyloid oligomers and amyloid fibrils by the multifunctional protein vitronectin

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Langen Ralf

2008-10-01

Full Text Available Abstract Background The multifunctional protein vitronectin is present within the deposits associated with Alzheimer disease (AD, age-related macular degeneration (AMD, atherosclerosis, systemic amyloidoses, and glomerulonephritis. The extent to which vitronectin contributes to amyloid formation within these plaques, which contain misfolded, amyloidogenic proteins, and the role of vitronectin in the pathophysiology of the aforementioned diseases is currently unknown. The investigation of vitronectin aggregation is significant since the formation of oligomeric and fibrillar structures are common features of amyloid proteins. Results We observed vitronectin immunoreactivity in senile plaques of AD brain, which exhibited overlap with the amyloid fibril-specific OC antibody, suggesting that vitronectin is deposited at sites of amyloid formation. Of particular interest is the growing body of evidence indicating that soluble nonfibrillar oligomers may be responsible for the development and progression of amyloid diseases. In this study we demonstrate that both plasma-purified and recombinant human vitronectin readily form spherical oligomers and typical amyloid fibrils. Vitronectin oligomers are toxic to cultured neuroblastoma and retinal pigment epithelium (RPE cells, possibly via a membrane-dependent mechanism, as they cause leakage of synthetic vesicles. Oligomer toxicity was attenuated in RPE cells by the anti-oligomer A11 antibody. Vitronectin fibrils contain a C-terminal protease-resistant fragment, which may approximate the core region of residues essential to amyloid formation. Conclusion These data reveal the propensity of vitronectin to behave as an amyloid protein and put forth the possibilities that accumulation of misfolded vitronectin may contribute to aggregate formation seen in age-related amyloid diseases.

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

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.

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.

Recombinant DNA derived monomeric insulin analogue: comparison with soluble human insulin in normal subjects.

Science.gov (United States)

Vora, J P; Owens, D R; Dolben, J; Atiea, J A; Dean, J D; Kang, S; Burch, A; Brange, J

1988-11-12

To compare the rate of absorption from subcutaneous tissue and the resulting hypoglycaemic effect of iodine-125 labelled soluble human insulin and a monomeric insulin analogue derived by recombinant DNA technology. Single blind randomised comparison of equimolar doses of 125I labelled soluble human insulin and insulin analogue. Study in normal people at a diabetes research unit and a university department of medical physics. Seven healthy male volunteers aged 20-39 not receiving any other drugs. After an overnight fast and a basal period of one hour two doses (0.05 and 0.1 U/kg) of 125I labelled soluble human insulin and insulin analogue were injected subcutaneously into the anterior abdominal wall on four separate days. To find a fast acting insulin for meal related requirements in insulin dependent diabetics. MEASUREMENTS and main results--Residual radioactivity at the injection site was measured continuously for the first two hours after injection of the 125I labelled preparations and thereafter for five minutes simultaneously with blood sampling. Frequent venous blood samples were obtained over six hours for determination of plasma immunoreactive insulin, insulin analogue, glucose, and glucagon values. Time to 50% of initial radioactivity at the injection site for the insulin analogue compared with soluble insulin was 61 v 135 minutes (p less than 0.05) with 0.05 U/kg and 67 v 145 minutes (p less than 0.001) with 0.1 U/kg. Concentrations in plasma increased faster after the insulin analogue compared with soluble insulin, resulting in higher plasma concentrations between 10 and 150 minutes (0.001 less than p less than 0.05) after 0.05 U/kg and between 40 and 360 minutes (0.001 less than p less than 0.05) after 0.1 U/kg. The hypoglycaemic response to insulin analogue was a plasma glucose nadir at 60 minutes with both doses compared with 90 and 120 minutes with soluble insulin at 0.5 and 0.1 U/kg respectively. The response of glucagon substantiated the earlier and

Screening of genetic parameters for soluble protein expression in Escherichia coli

DEFF Research Database (Denmark)

Vernet, Erik; Kotzsch, Alexander; Voldborg, Bjørn

2011-01-01

Soluble expression of proteins in a relevant form for functional and structural investigations still often remains a challenge. Although many biochemical factors are known to affect solubility, a thorough investigation of yield-limiting factors is normally not feasible in high-throughput efforts....... Here we present a screening strategy for expression of biomedically relevant proteins in Escherichia coli using a panel of six different genetic variations. These include engineered strains for rare codon supplementation, increased disulfide bond formation in the cytoplasm and novel vectors...... for secretion to the periplasm or culture medium. Combining these variants with expression construct truncations design, we report on parallel cloning and expression of more than 300 constructs representing 24 selected proteins; including full-length variants of human growth factors, interleukins and growth...

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.

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

Soluble polymorphic bank vole prion proteins induced by co-expression of quiescin sulfhydryl oxidase in E. coli and their aggregation behaviors.

Science.gov (United States)

Abskharon, Romany; Dang, Johnny; Elfarash, Ameer; Wang, Zerui; Shen, Pingping; Zou, Lewis S; Hassan, Sedky; Wang, Fei; Fujioka, Hisashi; Steyaert, Jan; Mulaj, Mentor; Surewicz, Witold K; Castilla, Joaquín; Wohlkonig, Alexandre; Zou, Wen-Quan

2017-10-04

The infectious prion protein (PrP Sc or prion) is derived from its cellular form (PrP C ) through a conformational transition in animal and human prion diseases. Studies have shown that the interspecies conversion of PrP C to PrP Sc is largely swayed by species barriers, which is mainly deciphered by the sequence and conformation of the proteins among species. However, the bank vole PrP C (BVPrP) is highly susceptible to PrP Sc from different species. Transgenic mice expressing BVPrP with the polymorphic isoleucine (109I) but methionine (109M) at residue 109 spontaneously develop prion disease. To explore the mechanism underlying the unique susceptibility and convertibility, we generated soluble BVPrP by co-expression of BVPrP with Quiescin sulfhydryl oxidase (QSOX) in Escherichia coli. Interestingly, rBVPrP-109M and rBVPrP-109I exhibited distinct seeded aggregation pathways and aggregate morphologies upon seeding of mouse recombinant PrP fibrils, as monitored by thioflavin T fluorescence and electron microscopy. Moreover, they displayed different aggregation behaviors induced by seeding of hamster and mouse prion strains under real-time quaking-induced conversion. Our results suggest that QSOX facilitates the formation of soluble prion protein and provide further evidence that the polymorphism at residue 109 of QSOX-induced BVPrP may be a determinant in mediating its distinct convertibility and susceptibility.

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.

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

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...

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

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.

Immunochemical analyses of soluble lens proteins in some marine fishes

Digital Repository Service at National Institute of Oceanography (India)

Menezes, M.R.

Soluble eye lens proteins of 10 fishes, belonging to the families Clupeidae, Hemirhamphidae, Lactaridae, Scombridae, Stromatidae, Psettodidae, Bothidae and Soleidae were studied by immunoelectrophoresis using the lens antiserum of Sardinella...

The evaluation of the factors that cause aggregation during recombinant expression in E. coli is simplified by the employment of an aggregation-sensitive reporter

Directory of Open Access Journals (Sweden)

Martinez Lucia

2006-09-01

Full Text Available Abstract Background The yields of soluble recombinant proteins expressed in bacteria are often low due to the tendency of the heterologous proteins to form aggregates. Therefore, aggregation reporters have been envisaged to simplify the comparison among different expression conditions and to speed up the identification of suitable protocols that improve the solubility. The probe we used is composed by an IbpAB promoter specifically activated by protein aggregates fused to a sequence coding the β-galactosidase, the activity of which becomes, therefore, indicative of the aggregation degree. Results The collected data show that the probe is reliable in terms of reproducibility inside a range of experimental conditions and faster and more sensitive than the analysis methods based on SDS-PAGE and successive western blot. The β-galactosidase probe was useful to identify which parameters could influence the aggregation of the model proteins and to set up an optimized expression protocol. The effect of growth temperature, induction modality, co-expression with molecular chaperones and addition of osmolytes on the accumulation of aggregates were evaluated following the β-galactosidase activity. Interestingly, a significant correlation was observed between estimated decreased aggregation and higher yields of soluble protein. We also compared a set of expression vectors with various regulative features and found that the single characteristics, like promoter, copy number or polymerase, were not relevant for controlling the recombinant protein aggregation whilst the crucial factor resulted being the total expression rate of the system. Conclusion The aggregation reporter used in our experiments represents a useful tool to evaluate the different factors that can be modulated to optimize a recombinant expression protocol. Furthermore, the rapid estimation of the aggregation degree enables to discriminate this from other causes responsible for scarce

Production and characterization of cowpea protein hydrolysate with optimum nitrogen solubility by enzymatic hydrolysis using pepsin.

Science.gov (United States)

Mune Mune, Martin Alain; Minka, Samuel René

2017-06-01

Cowpea is a source of low-cost and good nutritional quality protein for utilization in food formulations in replacement of animal proteins. Therefore it is necessary that cowpea protein exhibits good functionality, particularly protein solubility which affects the other functional properties. The objective of this study was to produce cowpea protein hydrolysate exhibiting optimum solubility by the adequate combination of hydrolysis parameters, namely time, solid/liquid ratio (SLR) and enzyme/substrate ratio (ESR), and to determine its functional properties and molecular characteristics. A Box-Behnken experimental design was used for the experiments, and a second-order polynomial to model the effects of hydrolysis time, SLR and ESR on the degree of hydrolysis and nitrogen solubility index. The optimum hydrolysis conditions of time 208.61 min, SLR 1/15 (w/w) and ESR 2.25% (w/w) yielded a nitrogen solubility of 75.71%. Protein breakdown and the peptide profile following enzymatic hydrolysis were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and size exclusion chromatography. Cowpea protein hydrolysate showed higher oil absorption capacity, emulsifying activity and foaming ability compared with the concentrate. The solubility of cowpea protein hydrolysate was adequately optimized by response surface methodology, and the hydrolysate showed adequate functionality for use in food. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Identification of immunogenic proteins and evaluation of four recombinant proteins as potential vaccine antigens from Vibrio anguillarum in flounder (Paralichthys olivaceus).

Science.gov (United States)

Xing, Jing; Xu, Hongsen; Wang, Yang; Tang, Xiaoqian; Sheng, Xiuzhen; Zhan, Wenbin

2017-05-31

Vibrio anguillarum is a severe bacterial pathogen that can infect a wide range of fish species. Identification of immunogenic proteins and development of vaccine are essential for disease prevention. In this study, immunogenic proteins were screened and identified from V. anguillarum, and then protective efficacy of the immunogenic proteins was evaluated. Immunogenic proteins in V. anguillarum whole cell were detected by Western blotting (WB) using immunized flounder (Paralichthys olivaceus) serum, and then identified by Mass spectrometry (MS). The recombinant proteins of four identified immunogenic proteins were produced and immunized to fish, and then percentages of surface membrane immunoglobulin-positive (sIg+) cells in peripheral blood lymphocytes (PBL), total antibodies, antibodies against V. anguillarum, antibodies against recombinant proteins and relative percent survival (RPS) were measured, respectively. The results showed that five immunogenic proteins, VAA, Groel, OmpU, PteF and SpK, were identified; their recombinant proteins, rOmpU, rGroel, rSpK and rVAA, could induce the proliferation of sIg+ cells in PBL and production of total antibodies, antibodies against V. anguillarum and antibodies against the recombinant proteins; their protection against V. anguillarum showed 64.86%, 72.97%, 21.62% and 78.38% RPS, respectively. The results revealed that the immunoproteomic technique using fish anti-V. anguillarum serum provided an efficient way to screen the immunogenic protein for vaccine antigen. Moreover, the rVAA, rGroel and rOmpU had potential to be vaccine candidates against V. anguillarum infection. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

Expression, purification and characterization of soluble red rooster laforin as a fusion protein in Escherichia coli.

Science.gov (United States)

Brewer, M Kathryn; Husodo, Satrio; Dukhande, Vikas V; Johnson, Mary Beth; Gentry, Matthew S

2014-04-02

The gene that encodes laforin, a dual-specificity phosphatase with a carbohydrate-binding module, is mutated in Lafora disease (LD). LD is an autosomal recessive, fatal progressive myoclonus epilepsy characterized by the intracellular buildup of insoluble, hyperphosphorylated glycogen-like particles, called Lafora bodies. Laforin dephosphorylates glycogen and other glucans in vitro, but the structural basis of its activity remains unknown. Recombinant human laforin when expressed in and purified from E. coli is largely insoluble and prone to aggregation and precipitation. Identification of a laforin ortholog that is more soluble and stable in vitro would circumvent this issue. In this study, we cloned multiple laforin orthologs, established a purification scheme for each, and tested their solubility and stability. Gallus gallus (Gg) laforin is more stable in vitro than human laforin, Gg-laforin is largely monomeric, and it possesses carbohydrate binding and phosphatase activity similar to human laforin. Gg-laforin is more soluble and stable than human laforin in vitro, and possesses similar activity as a glucan phosphatase. Therefore, it can be used to model human laforin in structure-function studies. We have established a protocol for purifying recombinant Gg-laforin in sufficient quantity for crystallographic and other biophysical analyses, in order to better understand the function of laforin and define the molecular mechanisms of Lafora disease.

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.

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...

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...

Biochemical characterization of soluble proteins in pecan [Carya illinoinensis (Wangenh.) K. Koch].

Science.gov (United States)

Venkatachalam, Mahesh; Roux, Kenneth H; Sathe, Shridhar K

2008-09-10

Pecans (cv. Desirable) contained approximately 10% protein on a dry weight basis. The minimum nitrogen solubility (5.9-7.5%) at 0.25-0.75 M trichloroacetic acid represented the nonprotein nitrogen. Among the solvents assessed for protein solubilization, 0.1 M NaOH was the most effective, while borate saline buffer (pH 8.45) was judged to be optimal for protein solubilization. The protein solubility was minimal in the pH range of 3-7 and significantly increased on either side of this pH range. Increasing the NaCl concentration from 0 to 4 M significantly improved ( approximately 8-fold increase) protein solubilization. Following Osborne protein fractionation, the alkali-soluble glutelin fraction (60.1%) accounted for a major portion of pecan proteins followed by globulin (31.5%), prolamin (3.4%), and albumin (1.5%), respectively. The majority of pecan polypeptides were in the molecular mass range of 12-66 kDa and in the pI range of 4.0-8.3. The pecan globulin fraction was characterized by the presence of several glycoprotein polypeptides. Lysine was the first limiting essential amino acid in the defatted flour, globulin, prolamin, and alkaline glutelin fractions. Leucine and tryptophan were the first limiting essential amino acids in albumin and acid glutelin fractions, respectively. Rabbit polyclonal antibodies detected a range of pecan polypeptides in the 12-60 kDa range, of which the globulin fraction contained the most reactive polypeptides.

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.

Factors Affecting the Binding of a Recombinant Heavy Metal-Binding Domain (CXXC motif Protein to Heavy Metals

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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.

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

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.

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)

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.

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.

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

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...

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

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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

Recombinant proteins in therapeutics: haemophilia treatment as an example

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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.

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...

Studies to Prevent Degradation of Recombinant Fc-Fusion Protein Expressed in Mammalian Cell Line and Protein Characterization

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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.

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.

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.

[Rapid expression and preparation of the recombinant fusion protein sTNFRII-gAD by adenovirus vector system].

Science.gov (United States)

Lu, Yue; Liu, Dan; Zhang, Xiaoren; Liu, Xuerong; Shen, Wei; Zheng, Gang; Liu, Yunfan; Dong, Xiaoyan; Wu, Xiaobing; Gao, Jimin

2011-08-01

We expressed and prepared the recombinant fusion protein sTNFRII-gAD consisted of soluble TNF receptor II and the globular domain of adiponectin by Adenovirus Vector System in mammalian BHK21c022 cells. First we used the adenovirus vector containing EGFP gene (rAd5-EGFP) to infect BHK21c022 cells at different MOI (from 0 to 1 000), and then evaluated their transduction efficiency and cytotoxicity. Similarly, we constructed the replication-deficient adenovirus type 5-sTNFRII-gAD (rAd5-sTNFRII-gAD). We collected the supernatants for Western blotting to determine the optimal MOI by comparing the expression levels of sTNFRII-gAD fusion protein, 48 h after the BHK21c022 cells were infected by rAd5-sTNFRII-gAD at different MOIs (from 0 to 1 000). Then, we chose rAd5-sTNFRII-gAD at MOI 100 to infect five bottles of BHK21c022 cells in 100 mL of serum-free chemically defined media 100 mL, harvested the supernatant every 48 h for 6 times, and condense and purify sTNFRII-gAD fusion protein by ammonium sulfate salt-out and size-exclusion chromatography, respectively. Finally, we analyzed anti-TNFalpha activity of sTNFRII-gAD fusion protein on L929 cells in vitro. The results showed that the number of BHK21c022 cells expressing EGFP protein was increased significantly with the increase of MOI. However, some cells died at MOI of 1 000 while there was no significant cytotoxicity at MOI from 0 to 100. Western blotting analysis showed that the more adenoviruses, the higher expression of sTNFRII-gAD fusion protein in the supernatant with the highest expression at MOI 1 000. We successfully obtained about 11 mg bioactive and purified sTNFRII-gAD fusion protein at last. The in vitro assay demonstrated that the sTNFRII-gAD fusion protein was potent to antagonize TNFalpha's cytotoxicity to L929 cells. Put together, we established a recombinant adenovirus vector/BHK21 cell expression system, characteristic of the efficient serum-free culture and easy scaling-up.

Comparative immunological evaluation of recombinant Salmonella Typhimurium strains expressing model antigens as live oral vaccines.

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Zheng, Song-yue; Yu, Bin; Zhang, Ke; Chen, Min; Hua, Yan-Hong; Yuan, Shuofeng; Watt, Rory M; Zheng, Bo-Jian; Yuen, Kwok-Yung; Huang, Jian-Dong

2012-09-26

Despite the development of various systems to generate live recombinant Salmonella Typhimurium vaccine strains, little work has been performed to systematically evaluate and compare their relative immunogenicity. Such information would provide invaluable guidance for the future rational design of live recombinant Salmonella oral vaccines. To compare vaccine strains encoded with different antigen delivery and expression strategies, a series of recombinant Salmonella Typhimurium strains were constructed that expressed either the enhanced green fluorescent protein (EGFP) or a fragment of the hemagglutinin (HA) protein from the H5N1 influenza virus, as model antigens. The antigens were expressed from the chromosome, from high or low-copy plasmids, or encoded on a eukaryotic expression plasmid. Antigens were targeted for expression in either the cytoplasm or the outer membrane. Combinations of strategies were employed to evaluate the efficacy of combined delivery/expression approaches. After investigating in vitro and in vivo antigen expression, growth and infection abilities; the immunogenicity of the constructed recombinant Salmonella strains was evaluated in mice. Using the soluble model antigen EGFP, our results indicated that vaccine strains with high and stable antigen expression exhibited high B cell responses, whilst eukaryotic expression or colonization with good construct stability was critical for T cell responses. For the insoluble model antigen HA, an outer membrane expression strategy induced better B cell and T cell responses than a cytoplasmic strategy. Most notably, the combination of two different expression strategies did not increase the immune response elicited. Through systematically evaluating and comparing the immunogenicity of the constructed recombinant Salmonella strains in mice, we identified their respective advantages and deleterious or synergistic effects. Different construction strategies were optimally-required for soluble versus

Common structural features of cholesterol binding sites in crystallized soluble proteins.

Science.gov (United States)

Bukiya, Anna N; Dopico, Alejandro M

2017-06-01

Cholesterol-protein interactions are essential for the architectural organization of cell membranes and for lipid metabolism. While cholesterol-sensing motifs in transmembrane proteins have been identified, little is known about cholesterol recognition by soluble proteins. We reviewed the structural characteristics of binding sites for cholesterol and cholesterol sulfate from crystallographic structures available in the Protein Data Bank. This analysis unveiled key features of cholesterol-binding sites that are present in either all or the majority of sites: i ) the cholesterol molecule is generally positioned between protein domains that have an organized secondary structure; ii ) the cholesterol hydroxyl/sulfo group is often partnered by Asn, Gln, and/or Tyr, while the hydrophobic part of cholesterol interacts with Leu, Ile, Val, and/or Phe; iii ) cholesterol hydrogen-bonding partners are often found on α-helices, while amino acids that interact with cholesterol's hydrophobic core have a slight preference for β-strands and secondary structure-lacking protein areas; iv ) the steroid's C21 and C26 constitute the "hot spots" most often seen for steroid-protein hydrophobic interactions; v ) common "cold spots" are C8-C10, C13, and C17, at which contacts with the proteins were not detected. Several common features we identified for soluble protein-steroid interaction appear evolutionarily conserved. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

Immobilized metal-affinity chromatography protein-recovery screening is predictive of crystallographic structure success

International Nuclear Information System (INIS)

Choi, Ryan; Kelley, Angela; Leibly, David; Nakazawa Hewitt, Stephen; Napuli, Alberto; Van Voorhis, Wesley

2011-01-01

An overview of the methods used for high-throughput cloning and protein-expression screening of SSGCID hexahistidine recombinant proteins is provided. It is demonstrated that screening for recombinant proteins that are highly recoverable from immobilized metal-affinity chromatography improves the likelihood that a protein will produce a structure. The recombinant expression of soluble proteins in Escherichia coli continues to be a major bottleneck in structural genomics. The establishment of reliable protocols for the performance of small-scale expression and solubility testing is an essential component of structural genomic pipelines. The SSGCID Protein Production Group at the University of Washington (UW-PPG) has developed a high-throughput screening (HTS) protocol for the measurement of protein recovery from immobilized metal-affinity chromatography (IMAC) which predicts successful purification of hexahistidine-tagged proteins. The protocol is based on manual transfer of samples using multichannel pipettors and 96-well plates and does not depend on the use of robotic platforms. This protocol has been applied to evaluate the expression and solubility of more than 4000 proteins expressed in E. coli. The UW-PPG also screens large-scale preparations for recovery from IMAC prior to purification. Analysis of these results show that our low-cost non-automated approach is a reliable method for the HTS demands typical of large structural genomic projects. This paper provides a detailed description of these protocols and statistical analysis of the SSGCID screening results. The results demonstrate that screening for proteins that yield high recovery after IMAC, both after small-scale and large-scale expression, improves the selection of proteins that can be successfully purified and will yield a crystal structure

Efficient soluble expression of disulfide bonded proteins in the cytoplasm of Escherichia coli in fed-batch fermentations on chemically defined minimal media.

Science.gov (United States)

Gąciarz, Anna; Khatri, Narendar Kumar; Velez-Suberbie, M Lourdes; Saaranen, Mirva J; Uchida, Yuko; Keshavarz-Moore, Eli; Ruddock, Lloyd W

2017-06-15

The production of recombinant proteins containing disulfide bonds in Escherichia coli is challenging. In most cases the protein of interest needs to be either targeted to the oxidizing periplasm or expressed in the cytoplasm in the form of inclusion bodies, then solubilized and re-folded in vitro. Both of these approaches have limitations. Previously we showed that soluble expression of disulfide bonded proteins in the cytoplasm of E. coli is possible at shake flask scale with a system, known as CyDisCo, which is based on co-expression of a protein of interest along with a sulfhydryl oxidase and a disulfide bond isomerase. With CyDisCo it is possible to produce disulfide bonded proteins in the presence of intact reducing pathways in the cytoplasm. Here we scaled up production of four disulfide bonded proteins to stirred tank bioreactors and achieved high cell densities and protein yields in glucose fed-batch fermentations, using an E. coli strain (BW25113) with the cytoplasmic reducing pathways intact. Even without process optimization production of purified human single chain IgA 1 antibody fragment reached 139 mg/L and hen avidin 71 mg/L, while purified yields of human growth hormone 1 and interleukin 6 were around 1 g/L. Preliminary results show that human growth hormone 1 was also efficiently produced in fermentations of W3110 strain and when glucose was replaced with glycerol as the carbon source. Our results show for the first time that efficient production of high yields of soluble disulfide bonded proteins in the cytoplasm of E. coli with the reducing pathways intact is feasible to scale-up to bioreactor cultivations on chemically defined minimal media.

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...

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

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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.

Synthesis of acid-soluble spore proteins by Bacillus subtilis.

OpenAIRE

Leventhal, J M; Chambliss, G H

1982-01-01

The major acid-soluble spore proteins (ASSPs) of Bacillus subtilis were detected by immunoprecipitation of radioactively labeled in vitro- and in vivo-synthesized proteins. ASSP synthesis in vivo began 2 h after the initiation of sporulation (t2) and reached its maximum rate at t7. This corresponded to the time of synthesis of mRNA that stimulated the maximum rate of ASSP synthesis in vitro. Under the set of conditions used in these experiments, protease synthesis began near t0, alkaline phos...

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

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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.

Polysome profiling of mAb producing CHO cell lines links translational control of cell proliferation and recombinant mRNA loading onto ribosomes with global and recombinant protein synthesis.

Science.gov (United States)

Godfrey, Charlotte L; Mead, Emma J; Daramola, Olalekan; Dunn, Sarah; Hatton, Diane; Field, Ray; Pettman, Gary; Smales, C Mark

2017-08-01

mRNA translation is a key process determining growth, proliferation and duration of a Chinese hamster ovary (CHO) cell culture and influences recombinant protein synthesis rate. During bioprocessing, CHO cells can experience stresses leading to reprogramming of translation and decreased global protein synthesis. Here we apply polysome profiling to determine reprogramming and translational capabilities in host and recombinant monoclonal antibody-producing (mAb) CHO cell lines during batch culture. Recombinant cell lines with the fastest cell specific growth rates were those with the highest global translational efficiency. However, total ribosomal capacity, determined from polysome profiles, did not relate to the fastest growing or highest producing mAb cell line, suggesting it is the ability to utilise available machinery that determines protein synthetic capacity. Cell lines with higher cell specific productivities tended to have elevated recombinant heavy chain transcript copy numbers, localised to the translationally active heavy polysomes. The highest titre cell line was that which sustained recombinant protein synthesis and maintained high recombinant transcript copy numbers in polysomes. Investigation of specific endogenous transcripts revealed a number that maintained or reprogrammed into heavy polysomes, identifying targets for potential cell engineering or those with 5' untranslated regions that might be utilised to enhance recombinant transcript translation. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Soluble Protein Analysis using a Compact Bench-top Flow Cytometer

Science.gov (United States)

Pappas, Dimitri; Kao, Shib-Hsin; Cyr, Johnathan

2004-01-01

Future space exploration missions will require analytical technology capable of providing both autonomous medical care to the crew and investigative capabilities to researchers. While several promising candidate technologies exist for further development, flow cytometry is an attractive technology as it offers both crew health (blood cell count, leukocyte differential, etc.) and a wide array of biochemistry and immunology assays. research settings, the application of this technique to soluble protein analysis is also possible. Proteomic beads using fluorescent dyes for optical encoding were used to monitor six cytokines simultaneously in cell medium of cell cultures in stationary and rotating cell culture systems. The results of this work demonstrate that a compact flow cytometer, such as a system proposed for space flight, can detect a variety of soluble proteins for crew health and biotechnology experiments during long-term missions.

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

Cloning, expression, and immunological characterization of recombinant Lolium perenne allergen Lol p II.

Science.gov (United States)

Sidoli, A; Tamborini, E; Giuntini, I; Levi, S; Volonté, G; Paini, C; De Lalla, C; Siccardi, A G; Baralle, F E; Galliani, S

1993-10-15

The molecular cloning of the cDNA encoding for an isoallergenic form of Lol p II, a major rye grass (Lolium perenne) pollen allergen, was performed by polymerase chain reaction amplification on mRNA extracted from pollen. The amino acid sequence derived from the cDNA was truncated by 4 and 5 residues at the NH2- and COOH-terminal ends, respectively, and differed only in one position from that previously reported. This cDNA was expressed in Escherichia coli by fusion to the carboxyl terminus of the human ferritin H-chain. The molecule was produced in high yields as a soluble protein and was easily purified. The protein retains the multimeric quaternary structure of ferritin, and it exposes on the surface the allergenic moiety, which can be recognized in Western blotting and in enzyme-linked immunosorbent assay experiments by specific IgE from allergic patients. The recombinant allergen was used to analyze the sera of 26 patients allergic to L. perenne compared with control sera. The results were in good agreement with the values obtained with the radioallergosorbent test assay. In addition, histamine release experiments in whole blood from an allergic patient and skin prick tests showed that the recombinant allergen retains some of the biological properties of the natural compound. These findings indicate that the availability of homogeneous recombinant allergens may be useful for the development of more specific diagnostic and therapeutic procedures. Moreover, this expression system may be of more general interest for producing large amounts of soluble protein domains in E. coli.

Expression of recombinant multi-coloured fluorescent antibodies in gor -/trxB- E. coli cytoplasm

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Markiv Anatoliy

2011-11-01

Full Text Available Abstract Background Antibody-fluorophore conjugates are invaluable reagents used in contemporary molecular cell biology for imaging, cell sorting and tracking intracellular events. However they suffer in some cases from batch to batch variation, partial loss of binding and susceptibility to photo-bleaching. In theory, these issues can all be addressed by using recombinant antibody fused directly to genetically encoded fluorescent reporters. However, single-chain fragment variable domains linked by long flexible linkers are themselves prone to disassociation and aggregation, and in some cases with isoelectric points incompatible with use in physiologically relevant milieu. Here we describe a general approach that permits fully functional intracellular production of a range of coloured fluorescent recombinant antibodies with optimally orientated VH/VL interfaces and isoelectric points compatible for use in physiological solutions at pH 7.4 with a binding site to fluorophore stoichiometry of 1:1. Results Here we report the design, assembly, intracellular bacterial production and purification of a panel of novel antibody fluorescent protein fusion constructs. The insertion of monomeric fluorescent protein derived from either Discosoma or Aequorea in-between the variable regions of anti-p185HER2-ECD antibody 4D5-8 resulted in optimal VH/VL interface interactions to create soluble coloured antibodies each with a single binding site, with isoelectric points of 6.5- 6. The fluorescent antibodies used in cell staining studies with SK-BR-3 cells retained the fluorophore properties and antibody specificity functions, whereas the conventional 4D5-8 single chain antibody with a (Gly4Ser3 linker precipitated at physiological pH 7.4. Conclusions This modular monomeric recombinant fluorescent antibody platform may be used to create a range of recombinant coloured antibody molecules for quantitative in situ, in vivo and ex vivo imaging, cell sorting and cell

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.

Semen quality and concentration of soluble proteins in the seminal plasma of Alpine bucks Semen quality and concentration of soluble proteins in the seminal plasma of Alpine bucks

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Simone Eliza Facione Guimarães

2010-06-01

Full Text Available It was aimed to study the in vitro seminal quality analyzed by complementary tests and to compare them with physical, morphological and biochemical aspects of male goat semen of the Alpine breed. This experiment took place at the Federal University of Viçosa, situated at 20º45’ S latitude and 42º51’ W longitude, Southwest of Brazil. It was done during the summer months of January and February, and three adult male goats of the Alpine breed were used in intensive conditions. The semen was collected by artificial vagina method. In all semen samples (45 ejaculates, after the physical and morphological analysis, the hiposmotic test was done. In 24 ejaculates, it were done thermo-resistance test, and in 21 ejaculates it were determined the concentration of total soluble proteins in seminal plasma. The male goats presented difference in the semen physical and morphological aspects, in the hiposmotic test and thermo-resistance test, but they did not presented difference in total soluble proteins concentration in seminal plasma. Results of the slow thermo-resistance test and hiposmotic test were positively correlated (r = 0.60. It was concluded, according to our results, that the concentration of total soluble proteins in seminal plasma can not be used as a parameter to predict the seminal quality of Alpine bucks.It was aimed to study the in vitro seminal quality analyzed by complementary tests and to compare them with physical, morphological and biochemical aspects of male goat semen of the Alpine breed. This experiment took place at the Federal University of Viçosa, situated at 20º45’ S latitude and 42º51’ W longitude, Southwest of Brazil. It was done during the summer months of January and February, and three adult male goats of the Alpine breed were used in intensive conditions. The semen was collected by artificial vagina method. In all semen samples (45 ejaculates, after the physical and morphological analysis, the hiposmotic test

Salt-soluble proteins from wheat-derived foodstuffs show lower allergenic potency than those from raw flour.

Science.gov (United States)

de Gregorio, Marta; Armentia, Alicia; Díaz-Perales, Araceli; Palacín, Arantxa; Dueñas-Laita, Antonio; Martín, Blanca; Salcedo, Gabriel; Sánchez-Monge, Rosa

2009-04-22

Salt-soluble proteins from wheat flour have been described as main allergens associated with both baker's asthma and food allergy. However, most studies have used raw flour as starting material, thus not considering potential changes in allergenic properties induced by the heat treatment and other industrial processing to produce wheat-derived foodstuffs. Salt extracts from different commercial wheat-derived products were obtained and their allergenic properties investigated by IgE-immunodetection, ELISA assays, and skin prick test. The IgE-binding capacity of salt-soluble proteins from commercial breads and cooked pastas was reduced around 50% compared with that of raw flour, the reduction being less dramatic in noncooked pastas and biscuits. Several wheat-derived foodstuffs showed major IgE-binding components of 20 and 35 kDa, identified as avenin-like and globulin proteins, respectively. These proteins, as well as most flour and bread salt-soluble proteins, were hydrolyzed when subjected to simulated gastrointestinal digestion. However, the digested products still exhibited a residual IgE-binding capacity. Therefore, processing of wheat flour to obtain derived foodstuffs decreases the IgE binding-capacity of the major salt-soluble wheat proteins. Moreover, simulated gastric fluid digestion further inactivates some heat-resistant IgE-binding proteins.

THE EFFECT OF THE PROTEIN SOLUBILITY OF FISH MEAL AND ...

African Journals Online (AJOL)

utilization of fish meal proteins was determined by a relationship between their solubility or apparent digestibility and the ... Data obtained from these trials should indicate what re- ..... 4 in mind it is thus obvious that the big variation which oc-.

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...

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...

ACCUMULATION OF RECOMBINANT FUSION PROTEIN – SECRETORY ANALOG OF Ag85B AND ESAT6 MYCOBACTERIUM TUBERCULOSIS PROTEINS – IN TRANSGENIC Lemna minor L. PLANTS

Directory of Open Access Journals (Sweden)

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.

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.

Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay.

Science.gov (United States)

Ni, Yan G; Yuan, Xiling; Newitt, John A; Peterson, Jon E; Gleason, Carol R; Haulenbeek, Jonathan; Santockyte, Rasa; Lafont, Virginie; Marsilio, Frank; Neely, Robert J; DeSilva, Binodh; Piccoli, Steven P

2015-07-01

Programmed death-1 (PD-1) protein is a co-inhibitory receptor which negatively regulates immune cell activation and permits tumors to evade normal immune defense. Anti-PD-1 antibodies have been shown to restore immune cell activation and effector function-an exciting breakthrough in cancer immunotherapy. Recent reports have documented a soluble form of PD-1 (sPD-1) in the circulation of normal and disease state individuals. A clinical assay to quantify sPD-1 would contribute to the understanding of sPD-1-function and facilitate the development of anti-PD-1 drugs. Here, we report the development and validation of a sPD-1 protein assay. The assay validation followed the framework for full validation of a biotherapeutic pharmacokinetic assay. A purified recombinant human PD-1 protein was characterized extensively and was identified as the assay reference material which mimics the endogenous analyte in structure and function. The lower limit of quantitation (LLOQ) was determined to be 100 pg/mL, with a dynamic range spanning three logs to 10,000 pg/mL. The intra- and inter-assay imprecision were ≤15%, and the assay bias (percent deviation) was ≤10%. Potential matrix effects were investigated in sera from both normal healthy volunteers and selected cancer patients. Bulk-prepared frozen standards and pre-coated Streptavidin plates were used in the assay to ensure consistency in assay performance over time. This assay appears to specifically measure total sPD-1 protein since the human anti-PD-1 antibody, nivolumab, and the endogenous ligands of PD-1 protein, PDL-1 and PDL-2, do not interfere with the assay.

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.

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.

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.

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.

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

Recombinant expression of homodimeric 660 kDa human thyroglobulin in soybean seeds: an alternative source of human thyroglobulin.

Science.gov (United States)

Powell, Rebecca; Hudson, Laura C; Lambirth, Kevin C; Luth, Diane; Wang, Kan; Bost, Kenneth L; Piller, Kenneth J

2011-07-01

Soybean seeds possess many qualities that make them ideal targets for the production of recombinant proteins. However, one quality often overlooked is their ability to stockpile large amounts of complex storage proteins. Because of this characteristic, we hypothesized that soybean seeds would support recombinant expression of large and complex proteins that are currently difficult or impossible to express using traditional plant and non-plant-based host systems. To test this hypothesis, we transformed soybeans with a synthetic gene encoding human thyroglobulin (hTG)-a 660 kDa homodimeric protein that is widely used in the diagnostic industry for screening and detection of thyroid disease. In the absence of a recombinant system that can produce recombinant hTG, research and diagnostic grade hTG continues to be purified from cadaver and surgically removed thyroid tissue. These less-than-ideal tissue sources lack uniform glycosylation and iodination and therefore introduce variability when purified hTG is used in sensitive ELISA screens. In this study, we report the successful expression of recombinant hTG in soybean seeds. Authenticity of the soy-derived protein was demonstrated using commercial ELISA kits developed specifically for the detection of hTG in patient sera. Western analyses and gel filtration chromatography demonstrated that recombinant hTG and thyroid-purified hTG are biologically similar with respect to size, mass, charge and subunit interaction. The recombinant protein was stable over three generations and accumulated to ~1.5% of total soluble seed protein. These results support our hypothesis that soybeans represent a practical alternative to traditional host systems for the expression of large and complex proteins.

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.

Expression of human DNA polymerase β in Escherichia coli and characterization of the recombinant enzyme

International Nuclear Information System (INIS)

Abbotts, J.; SenGupta, D.N.; Zmudzka, B.; Widen, S.G.; Notario, V.; Wilson, S.H.

1988-01-01

The coding region of a human β-polymerase cDNA, predicting a 335 amino acid protein, was subcloned in the Escherichia coli expression plasmid pRC23. After induction of transformed cells, the crude soluble extract was found to contain a new protein immunoreactive with β-polymerase antibody and corresponding in size to the protein deduced from the cDNA. This protein was purified in a yield of 1-2 mg/50 g of cells. The recombinant protein had about the same DNA polymerase specific activity as β-polymerase purified from mammalian tissues, and template-primer specificity and immunological properties of the recombinant polymerase were similar to those of natural β-polymerases. The purified enzyme was free of nuclease activity. The authors studied detailed catalytic properties of the recombinant β-polymerase using defined template-primer systems. The results indicate that this β-polymerase is essentially identical with natural β-polymerases. The recombinant enzyme is distributive in mode of synthesis and is capable of detecting changes in the integrity of the single-stranded template, such as methylated bases and a double-stranded region. The enzyme recognizes a template region four to seven bases downstream of the primer 3' end and utilizes alternative primers if this downstream template region is double stranded. The enzyme is unable to synthesize past methylated bases N 3 -methyl-dT or O 6 -methyl-dG

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

Rehosting of Bacterial Chaperones for High-Quality Protein Production▿

Science.gov (United States)

Martínez-Alonso, Mónica; Toledo-Rubio, Verónica; Noad, Rob; Unzueta, Ugutz; Ferrer-Miralles, Neus; Roy, Polly; Villaverde, Antonio

2009-01-01

Coproduction of DnaK/DnaJ in Escherichia coli enhances solubility but promotes proteolytic degradation of their substrates, minimizing the yield of unstable polypeptides. Higher eukaryotes have orthologs of DnaK/DnaJ but lack the linked bacterial proteolytic system. By coexpression of DnaK and DnaJ in insect cells with inherently misfolding-prone recombinant proteins, we demonstrate simultaneous improvement of soluble protein yield and quality and proteolytic stability. Thus, undesired side effects of bacterial folding modulators can be avoided by appropriate rehosting in heterologous cell expression systems. PMID:19820142

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.

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)

Soluble multimer of recombinant endostatin expressed in E. coli has anti-angiogenesis activity

International Nuclear Information System (INIS)

Wei Dongmei; Gao Yan; Cao Xiangrong; Zhu Nianchun; Liang Jianfu; Xie Weiping; Zhen Mingying; Zhu Minsheng

2006-01-01

The bioactivity, refolding, and multimer formation of endostatin, particularly of recombinant endostatin produced from bacteria, are proved challenging for clinical application. In order to determine the biological activity of recombinant endostatin multimer, first, we expressed endostatin in Escherichia coli and purified it with ion-exchange chromatography. The purified active protein could elicit multimer formation spontaneously, but still has comparable activity. Aim to determine the anti-angiogenic activity of multimer endostatin, by use of RP-HPLC, we then successfully separated endostatin monomer and multimer for subjecting to anti-angiogenesis assay. The results from CAM (chorioallantoic membrane) inhibition assay showed that both monomer and multimer suppressed CAM vascularization significantly. At the dosage of 0.8 μg, inhibition rates of multimeric and monomeric proteins were about 58% and 38%, respectively. Multimeric endostatin exerted a higher activity than monomeric endostatin (p 0.05), although both of them show a high inhibition effect in contrast to control. The results from HUVEC proliferation assay also showed similar effects at dosages of 0.6 and 1.6 μg/ml, multimer exerted a higher activity on inhibition of HUVEC proliferation comparing with monomer (p < 0.05). In conclusion, our results suggest that endostatin multimer has a comparable or higher bioactivity and multimerization will not affect its bioactivity, implying that endostatin activity is insensitive to structure conformation contributed by disulfide bonds

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.

A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production

Directory of Open Access Journals (Sweden)

Bottomley Stephen P

2006-03-01

Full Text Available Abstract Background In the past few years, both automated and manual high-throughput protein expression and purification has become an accessible means to rapidly screen and produce soluble proteins for structural and functional studies. However, many of the commercial vectors encoding different solubility tags require different cloning and purification steps for each vector, considerably slowing down expression screening. We have developed a set of E. coli expression vectors with different solubility tags that allow for parallel cloning from a single PCR product and can be purified using the same protocol. Results The set of E. coli expression vectors, encode for either a hexa-histidine tag or the three most commonly used solubility tags (GST, MBP, NusA and all with an N-terminal hexa-histidine sequence. The result is two-fold: the His-tag facilitates purification by immobilised metal affinity chromatography, whilst the fusion domains act primarily as solubility aids during expression, in addition to providing an optional purification step. We have also incorporated a TEV recognition sequence following the solubility tag domain, which allows for highly specific cleavage (using TEV protease of the fusion protein to yield native protein. These vectors are also designed for ligation-independent cloning and they possess a high-level expressing T7 promoter, which is suitable for auto-induction. To validate our vector system, we have cloned four different genes and also one gene into all four vectors and used small-scale expression and purification techniques. We demonstrate that the vectors are capable of high levels of expression and that efficient screening of new proteins can be readily achieved at the laboratory level. Conclusion The result is a set of four rationally designed vectors, which can be used for streamlined cloning, expression and purification of target proteins in the laboratory and have the potential for being adaptable to a high

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.

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...

Systematic high-yield production of human secreted proteins in Escherichia coli

International Nuclear Information System (INIS)

Dai Xueyu; Chen Qiang; Lian Min; Zhou Yanfeng; Zhou Mo; Lu Shanyun; Chen Yunjia; Luo Jingchu; Gu Xiaocheng; Jiang Ying; Luo Ming; Zheng Xiaofeng

2005-01-01

Human secreted proteins play a very important role in signal transduction. In order to study all potential secreted proteins identified from the human genome sequence, systematic production of large amounts of biologically active secreted proteins is a prerequisite. We selected 25 novel genes as a trial case for establishing a reliable expression system to produce active human secreted proteins in Escherichia coli. Expression of proteins with or without signal peptides was examined and compared in E. coli strains. The results indicated that deletion of signal peptides, to a certain extent, can improve the expression of these proteins and their solubilities. More importantly, under expression conditions such as induction temperature, N-terminus fusion peptides need to be optimized in order to express adequate amounts of soluble proteins. These recombinant proteins were characterized as well-folded proteins. This system enables us to rapidly obtain soluble and highly purified human secreted proteins for further functional studies

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...

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.

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...

Recombinant expression of a functional myo-inositol-1-phosphate synthase (MIPS) in Mycobacterium smegmatis.

Science.gov (United States)

Huang, Xinyi; Hernick, Marcy

2015-10-01

Myo-inositol-1-phosphate synthase (MIPS, E.C. 5.5.1.4) catalyzes the first step in inositol production-the conversion of glucose-6-phosphate (Glc-6P) to myo-inositol-1-phosphate. While the three dimensional structure of MIPS from Mycobacterium tuberculosis has been solved, biochemical studies examining the in vitro activity have not been reported to date. Herein we report the in vitro activity of mycobacterial MIPS expressed in E. coli and Mycobacterium smegmatis. Recombinant expression in E. coli yields a soluble protein capable of binding the NAD(+) cofactor; however, it has no significant activity with the Glc-6P substrate. In contrast, recombinant expression in M. smegmatis mc(2)4517 yields a functionally active protein. Examination of structural data suggests that MtMIPS expressed in E. coli adopts a fold that is missing a key helix containing two critical (conserved) Lys side chains, which likely explains the inability of the E. coli expressed protein to bind and turnover the Glc-6P substrate. Recombinant expression in M. smegmatis may yield a protein that adopts a fold in which this key helix is formed enabling proper positioning of important side chains, thereby allowing for Glc-6P substrate binding and turnover. Detailed mechanistic studies may be feasible following optimization of the recombinant MIPS expression protocol in M. smegmatis.

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

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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.

Recombinant Vaccinia Viruses Coding Transgenes of Apoptosis-Inducing Proteins Enhance Apoptosis But Not Immunogenicity of Infected Tumor Cells

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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

Tissue-engineered human bioartificial muscles expressing a foreign recombinant protein for gene therapy

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Powell, C.; Shansky, J.; Del Tatto, M.; Forman, D. E.; Hennessey, J.; Sullivan, K.; Zielinski, B. A.; Vandenburgh, H. H.

1999-01-01

Murine skeletal muscle cells transduced with foreign genes and tissue engineered in vitro into bioartificial muscles (BAMs) are capable of long-term delivery of soluble growth factors when implanted into syngeneic mice (Vandenburgh et al., 1996b). With the goal of developing a therapeutic cell-based protein delivery system for humans, similar genetic tissue-engineering techniques were designed for human skeletal muscle stem cells. Stem cell myoblasts were isolated, cloned, and expanded in vitro from biopsied healthy adult (mean age, 42 +/- 2 years), and elderly congestive heart failure patient (mean age, 76 +/- 1 years) skeletal muscle. Total cell yield varied widely between biopsies (50 to 672 per 100 mg of tissue, N = 10), but was not significantly different between the two patient groups. Percent myoblasts per biopsy (73 +/- 6%), number of myoblast doublings prior to senescence in vitro (37 +/- 2), and myoblast doubling time (27 +/- 1 hr) were also not significantly different between the two patient groups. Fusion kinetics of the myoblasts were similar for the two groups after 20-22 doublings (74 +/- 2% myoblast fusion) when the biopsy samples had been expanded to 1 to 2 billion muscle cells, a number acceptable for human gene therapy use. The myoblasts from the two groups could be equally transduced ex vivo with replication-deficient retroviral expression vectors to secrete 0.5 to 2 microg of a foreign protein (recombinant human growth hormone, rhGH)/10(6) cells/day, and tissue engineered into human BAMs containing parallel arrays of differentiated, postmitotic myofibers. This work suggests that autologous human skeletal myoblasts from a potential patient population can be isolated, genetically modified to secrete foreign proteins, and tissue engineered into implantable living protein secretory devices for therapeutic use.

Modulating secretory pathway pH by proton channel co-expression can increase recombinant protein stability in plants.

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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.

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...

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.

Evaluation of a competitive enzyme-linked immunosorbent assay for measurements of soluble HLA-G protein

DEFF Research Database (Denmark)

Rasmussen, M; Dahl, M; Buus, S

2014-01-01

. We report a novel method, a competitive immunoassay, for measuring HLA-G5/sHLA-G1 in biological fluids. The sHLA-G immunoassay is based upon a competitive enzyme-linked immunosorbent assay (ELISA) principle. It includes a recombinant sHLA-G1 protein in complex with β2-microglobulin and a peptide...... as a standard, biotinylated recombinant sHLA-G1 as an indicator, and the MEM-G/9 anti-HLA-G monoclonal antibody (mAb) as the capture antibody. The specificity and sensitivity of the assay were evaluated. Testing with different recombinant HLA class I proteins and different anti-HLA class I mAbs showed....../ml. An intra-assay coefficient of variation (CV) of 15.5% at 88 ng/ml and an inter-assay CV of 23.1% at 39 ng/ml were determined. An assay based on the competitive sHLA-G ELISA may be important for measurements of sHLA-G proteins in several conditions: assisted reproduction, organ transplantation, cancer...

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

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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.

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

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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.

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.

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.

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

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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.

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.

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.

High quality protein microarray using in situ protein purification

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Fleischmann Robert D

2009-08-01

protein solubility and denaturation problems caused by buffer exchange steps and freeze-thaw cycles, which are associated with resin-based purification, intermittent protein storage and deposition on microarrays. Conclusion An optimized platform for in situ protein purification on microarray slides using His-tagged recombinant proteins is a desirable tool for the screening of novel protein functions and protein-protein interactions. In the context of immunoproteomics, such protein microarrays are complimentary to approaches using non-recombinant methods to discover and characterize bacterial antigens.

Stable Plastid Transformation for High-Level Recombinant Protein Expression: Promises and Challenges

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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.

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

Anchoring tick salivary anti-complement proteins IRAC I and IRAC II to membrane increases their immunogenicity.

Science.gov (United States)

Gillet, Laurent; Schroeder, Hélène; Mast, Jan; Thirion, Muriel; Renauld, Jean-Christophe; Dewals, Benjamin; Vanderplasschen, Alain

2009-01-01

Tick salivary proteins are promising targets for the development of anti-tick vaccines. Recently, we described two paralogous anti-complement proteins, called Ixodes ricinus anti-complement (IRAC) proteins I and II, that are co-expressed in tick I. ricinus salivary glands. However, our previous attempts to immunize rabbits against IRAC via infection with recombinant Bovine herpesvirus 4 (BoHV-4) vectors invariably failed although both recombinants expressed high levels of functional IRAC proteins in vitro. As IRAC are soluble monovalent antigens, one of the possible explanations is that monovalent ligation of the B-cell receptor induces receptor activation but fails to promote antigen presentation, a phenomenon that is thought to induce a state of B-cell tolerance. In the present study, we tried to increase IRAC immunogenicity by expressing them as oligovalent antigens. To this end, IRAC were fused to membrane anchors and BoHV-4 vectors expressing these recombinant forms were produced. The immunization potentials of recombinant viruses expressing either secreted or transmembrane IRAC proteins were then compared. While the former did not induce a detectable immune response against IRAC, the latter led to high titres of anti-IRAC antibodies that only marginally affected tick blood feeding. All together, the data presented in this study demonstrate that the immunogenicity of a soluble antigen can be greatly improved by anchoring it in membrane.

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.

Photodamaging mechanism of the eye structure: UV effect on soluble proteins of the lens

International Nuclear Information System (INIS)

Korkhmazyan, M.M.; Fedorovich, I.B.; Ostrovskij, M.A.

1983-01-01

Damaging effect of UV-radiation on soluble proteins of bull lens has been studied. Irradiation results in lens proteins growing yellow, new absorption bands with the maxima 245 and 305 nm appear. It is shown that during photodamage oxidation of SH-groups takes place and protein aggregates are formed

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

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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.

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.

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.

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...

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...

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.

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.

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

TROUBLESHOOTING IN EXPRESSION AND PURIFICATION OF RECOMBINANT SEVERE ACUTE RESPIRATORY SYNDROME-ASSOCIATED CORONAVIRUS NUCLEOCAPSID PROTEIN IN Escherichia coli BL21

Directory of Open Access Journals (Sweden)

Budiman Bela

2010-11-01

Full Text Available Considering importance of N protein for study of viral pathogenesis or development of immunodiagnostic assay, wereported effects of several conditions on purity and homogeneity of recombinant SARS-CoV N protein expressed in E.coli BL21. The SARS-CoV N gene was reverse transcribed and amplified by the reverse transcription-polymerase chainreaction (RT-PCR technique. The amplicons were cloned into pGEX-6P1 and followed by subcloning of the targetgene into pQE-80L. After inserting the recombinant plasmid (pQE80-N into E. coli, the recombinant protein (6 x Histag-N protein fusion was expressed by inducing the bacterial cells with 0.1-0.5 mM isopropyl-1-thio-Dgalactopyranoside(IPTG for 1-5 h. The protein recombinant were extracted from the bacterial cells by NTT buffercontaining 0-20 mM imidazol, and followed by Ni-NTA affinity resin purification. The results showed that induction ofE. coli BL21 with 0.2 mM IPTG for 4 h and followed with lysis of bacterial cells in NTT buffer containing 10 mMimidazol were optimal conditions to obtain the pure recombinant SARS-CoV N protein.

Benzoate-mediated changes on expression profile of soluble proteins in Serratia sp. DS001.

Science.gov (United States)

Pandeeti, E V P; Chinnaboina, M R; Siddavattam, D

2009-05-01

To assess differences in protein expression profile associated with shift in carbon source from succinate to benzoate in Serratia sp. DS001 using a proteomics approach. A basic proteome map was generated for the soluble proteins extracted from Serratia sp. DS001 grown in succinate and benzoate. The differently and differentially expressed proteins were identified using ImageMaster 2D Platinum software (GE Healthcare). The identity of the proteins was determined by employing MS or MS/MS. Important enzymes such as Catechol 1,2 dioxygenase and transcriptional regulators that belong to the LysR superfamily were identified. Nearly 70 proteins were found to be differentially expressed when benzoate was used as carbon source. Based on the protein identity and degradation products generated from benzoate it is found that ortho pathway is operational in Serratia sp. DS001. Expression profile of the soluble proteins associated with shift in carbon source was mapped. The study also elucidates degradation pathway of benzoate in Serratia sp. DS001 by correlating the proteomics data with the catabolites of benzoate.

Properties of spores of Bacillus subtilis strains which lack the major small, acid-soluble protein

International Nuclear Information System (INIS)

Hackett, R.H.; Setlow, P.

1988-01-01

Bacillus subtilis strains containing a deletion in the gene coding for the major small, acid-soluble, spore protein (SASP-gamma) grew and sporulated, and their spores initiated germination normally, but outgrowth of SASP-gamma- spores was significantly slower than that of wild-type spores. The absence of SASP-gamma had no effect on spore protoplast density or spore resistance to heat or radiation. Consequently, SASP-gamma has a different function in spores than do the other major small, acid-soluble proteins

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.

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.

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.

Improved expression of recombinant plant-made hEGF.

Science.gov (United States)

Thomas, David Rhys; Walmsley, Amanda Maree

2014-11-01

The yield of recombinant hEGF was increased approximately tenfold through a range of optimisations. Further, the recombinant protein was found to have biological activity comparable to commercial hEGF. Human epidermal growth factor (hEGF) is a powerful mitogen that can enhance the healing of a wide range of injuries, including burns, cuts, diabetic ulcers and gastric ulcers. However, despite its clinical value, hEGF is only consistently used for the treatment of chronic diabetic ulcers due to its high cost. In this study, hEGF was transiently expressed in Nicotiana benthamiana plants and targeted to the apoplast, ER and vacuole. Several other approaches were also included in a stepwise fashion to identify the optimal conditions for the expression of recombinant hEGF. Expression was found to be highest in the vacuole, while targeting hEGF to the ER caused a decrease in total soluble protein (TSP). Using a codon optimised sequence was found to increase vacuolar targeted hEGF yield by ~34 %, while it was unable to increase the yield of ER targeted hEGF. The use of the P19 silencing inhibitor was able to further increase expression by over threefold, and using 5-week-old plants significantly increased expression compared to 4- or 6-week-old-plants. The combined effect of these optimisations increased expression tenfold over the initial apoplast targeted construct to an average yield of 6.24 % of TSP. The plant-made hEGF was then shown to be equivalent to commercial E. coli derived hEGF in its ability to promote the proliferation of mouse keratinocytes. This study supports the potential for plants to be used for the commercial production of hEGF, and identifies a potential limitation for the further improvement of recombinant protein yields.

Soluble lens protein polymorphism in flying fishes from the central Arabian Sea

Digital Repository Service at National Institute of Oceanography (India)

Menezes, M.R.

Soluble eye lens nuclei proteins of flying-fishes were studied by cellogel electrophoresis. Three distinct patterns characterized by the number of bands, mobility and staining intensity were observed. Morphological studies of these fishes showed...

Recombinant Salivary Proteins of Phlebotomus orientalis are Suitable Antigens to Measure Exposure of Domestic Animals to Sand Fly Bites.

Science.gov (United States)

Sima, Michal; Ferencova, Blanka; Warburg, Alon; Rohousova, Iva; Volf, Petr

2016-03-01

Certain salivary proteins of phlebotomine sand flies injected into the host skin during blood-feeding are highly antigenic and elicit strong antibody-mediated immune responses in repeatedly-exposed hosts. These antibodies can be measured by enzyme-linked immuno sorbent assays (ELISAs) using salivary gland homogenates (SGHs) as the source of antigens and serve as a markers for exposure to biting sand flies. Large-scale screening for anti-sand fly saliva antibodies requires replacement of SGH with recombinant salivary proteins. In East Africa, Phlebotomus orientalis is the main vector of Leishmania donovani, a trypanosomatid parasite causing visceral leishmaniasis. We tested recombinant salivary proteins derived from Ph. orientalis saliva to study exposure of domestic animals to this sand fly species. Antigenic salivary proteins from Ph. orientalis were identified by immunoblot and mass spectrometry. Recombinant apyrase rPorSP15, yellow-related protein rPorSP24, ParSP25-like protein rPorSP65, D7-related protein rPorSP67, and antigen 5-related protein rPorSP76 were tested using ELISA with sera of domestic animals from L. donovani foci in Ethiopia where Ph. orientalis is present. Our results highlighted recombinant yellow-related protein rPorSP24 as the most promising antigen, displaying a high positive correlation coefficient as well as good sensitivity and specificity when compared to SGH. This recombinant protein was the most suitable one for testing sera of dogs, sheep, and goats. In addition, a different antigen, rPorSP65 was found efficacious for testing canine sera. Recombinant salivary proteins of Ph. orientalis, specifically rPorSP24, were shown to successfully substitute SGH in serological experiments to measure exposure of domestic animals to Ph. orientalis, the vector of L. donovani. The results suggest that rPorSP24 might be a suitable antigen for detecting anti-Ph. orientalis antibody-mediated reactions also in other host species.

Prefoldin Promotes Proteasomal Degradation of Cytosolic Proteins with Missense Mutations by Maintaining Substrate Solubility.

Directory of Open Access Journals (Sweden)

Sophie A Comyn

2016-07-01

Full Text Available Misfolded proteins challenge the ability of cells to maintain protein homeostasis and can accumulate into toxic protein aggregates. As a consequence, cells have adopted a number of protein quality control pathways to prevent protein aggregation, promote protein folding, and target terminally misfolded proteins for degradation. In this study, we employed a thermosensitive allele of the yeast Guk1 guanylate kinase as a model misfolded protein to investigate degradative protein quality control pathways. We performed a flow cytometry based screen to identify factors that promote proteasomal degradation of proteins misfolded as the result of missense mutations. In addition to the E3 ubiquitin ligase Ubr1, we identified the prefoldin chaperone subunit Gim3 as an important quality control factor. Whereas the absence of GIM3 did not impair proteasomal function or the ubiquitination of the model substrate, it led to the accumulation of the poorly soluble model substrate in cellular inclusions that was accompanied by delayed degradation. We found that Gim3 interacted with the Guk1 mutant allele and propose that prefoldin promotes the degradation of the unstable model substrate by maintaining the solubility of the misfolded protein. We also demonstrated that in addition to the Guk1 mutant, prefoldin can stabilize other misfolded cytosolic proteins containing missense mutations.

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.

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....

Fcγ1 fragment of IgG1 as a powerful affinity tag in recombinant Fc-fusion proteins: immunological, biochemical and therapeutic properties.

Science.gov (United States)

Soleimanpour, Saman; Hassannia, Tahereh; Motiee, Mahdieh; Amini, Abbas Ali; Rezaee, S A R

2017-05-01

Affinity tags are vital tools for the production of high-throughput recombinant proteins. Several affinity tags, such as the hexahistidine tag, maltose-binding protein, streptavidin-binding peptide tag, calmodulin-binding peptide, c-Myc tag, glutathione S-transferase and FLAG tag, have been introduced for recombinant protein production. The fragment crystallizable (Fc) domain of the IgG1 antibody is one of the useful affinity tags that can facilitate detection, purification and localization of proteins and can improve the immunogenicity, modulatory effects, physicochemical and pharmaceutical properties of proteins. Fcγ recombinant forms a group of recombinant proteins called Fc-fusion proteins (FFPs). FFPs are widely used in drug discovery, drug delivery, vaccine design and experimental research on receptor-ligand interactions. These fusion proteins have become successful alternatives to monoclonal antibodies for drug developments. In this review, the physicochemical, biochemical, immunological, pharmaceutical and therapeutic properties of recombinant FFPs were discussed as a new generation of bioengineering strategies.

A lower isoelectric point increases signal sequence-mediated secretion of recombinant proteins through a bacterial ABC transporter.

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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.

Membrane fusion between baculovirus budded virus-enveloped particles and giant liposomes generated using a droplet-transfer method for the incorporation of recombinant membrane proteins.