Strategies to Optimize Protein Expression in E. coli

Dana M. Francis1, Rebecca Page1

1 Brown University, Providence, Rhode Island
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 5.24
DOI:  10.1002/0471140864.ps0524s61
Online Posting Date:  August, 2010
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Abstract

Recombinant protein expression in Escherichia coli (E. coli) is simple, fast, inexpensive, and robust, with the expressed protein comprising up to 50 percent of the total cellular protein. However, it also has disadvantages. For example, the rapidity of bacterial protein expression often results in unfolded/misfolded proteins, especially for heterologous proteins that require longer times and/or molecular chaperones to fold correctly. In addition, the highly reductive environment of the bacterial cytosol and the inability of E. coli to perform several eukaryotic post‐translational modifications results in the insoluble expression of proteins that require these modifications for folding and activity. Fortunately, multiple, novel reagents and techniques have been developed that allow for the efficient, soluble production of a diverse range of heterologous proteins in E. coli. This overview describes variables at each stage of a protein expression experiment that can influence solubility and offers a summary of strategies used to optimize soluble expression in E. coli. Curr. Protoc. Protein Sci. 61:5.24.1‐5.24.29. © 2010 by John Wiley & Sons, Inc.

Keywords: protein expression; E. coli; fusion proteins; proteases; heterologous protein; purification tags; expression tags; expression strains and vectors; folded protein; active protein

     
 
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Table of Contents

  • Introduction
  • I. Representative Protocol for Expressing Proteins in Bacteria
  • II. Properties of the Gene and Protein that Influence Expression and Solubility
  • III. Properties of the Vector that Influence Expression and Solubility
  • IV. E. coli Host Strains that aid Expression of Heterologous Proteins
  • V. The Solubility of Proteins can be Improved by Changing Expression Conditions
  • VI. Enhancing Solubility by Coexpression with Other Proteins
  • Anticipated Results and time Considerations
  • Acknowledgments
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

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Literature Cited

Literature Cited
   Amada, K., Yohda, M., Odaka, M., Endo, I., Ishii, N., Taguchi, H., and Yoshida, M. 1995. Molecular‐cloning, expression, and characterization of Chaperonin‐60 and Chaperonin‐10 from a thermophilic bacterium, Thermus‐Thermophilus Hb8. J. Biochem. 118:347–354.
   Amann, E., Brosius, J., and Ptashne, M. 1983. Vectors bearing a hybrid Trp‐Lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene 25:167‐178.
   Armstrong, R.N. 1997. Structure, catalytic mechanism, and evolution of the glutathione transferases. Chem. Res. Toxicol. 10:2‐18.
   Arnold, K., Bordoli, L., Kopp, J., and Schwede, T. 2006. The SWISS‐MODEL workspace: A web‐based environment for protein structure homology modelling. Bioinformatics 22:195‐201.
   Ayling, A. and Baneyx, F. 1996. Influence of the GroE molecular chaperone machine on the in vitro refolding of Escherichia coli beta‐galactosidase. Protein Sci. 5:478‐487.
   Baneyx, F. and Mujacic, M. 2004. Recombinant protein folding and misfolding in Escherichia coli. Nat. Biotechnol. 22:1399‐1408.
   Baneyx, F. and Palumbo, J.L. 2003. Improving heterologous protein folding via molecular chaperone and foldase co‐expression. Methods Mol. Biol. 205:171‐197.
   Bao, W.J., Gao, Y.G., Chang, Y.G., Zhang, T.Y., Lin, X.J., Yan, X.Z., and Hu, H.Y. 2006. Highly efficient expression and purification system of small‐size protein domains in Escherichia coli for biochemical characterization. Protein Expr. Purif. 47:599‐606.
   Bessette, P.H., Aslund, F., Beckwith, J., and Georgiou, G. 1999. Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm. Proc. Natl. Acad. Sci. U.S.A. 96:13703‐13708.
   Brosius, J., Erfle, M., and Storella, J. 1985. Spacing of the −10 and −35 regions in the tac promoter. Effect on its in vivo activity. J. Biol. Chem. 260:3539‐3541.
   Brown, B.L., Hadley, M., and Page, R. 2008. Heterologous high‐level E. coli expression, purification and biophysical characterization of the spine‐associated RapGAP (SPAR) PDZ domain. Protein Expr. Purif. 62:9‐14.
   Brown, B.L., Grigoriu, S., Kim, Y., Arruda, J.M., Davenport, A., Wood, T.K., Peti, W., and Page, R. 2009. Three dimensional structure of the MqsR:MqsA complex: A novel TA pair comprised of a toxin homologous to RelE and an antitoxin with unique properties. PLoS Pathog. 5:e100706.
   Burgess‐Brown, N.A., Sharma, S., Sobott, F., Loenarz, C., Oppermann, U., and Gileadi, O. 2008. Codon optimization can improve expression of human genes in Escherichia coli: A multi‐gene study. Protein Expr. Purif. 59:94‐102.
   Busso, D., Delagoutte‐Busso, B., and Moras, D. 2005. Construction of a set Gateway‐based destination vectors for high‐throughput cloning and expression screening in Escherichia coli. Anal. Biochem. 343:313‐321.
   Butt, T.R., Jonnalagadda, S., Monia, B.P., Sternberg, E.J., Marsh, J.A., Stadel, J.M., Ecker, D.J., and Crooke, S.T. 1989. Ubiquitin fusion augments the yield of cloned gene‐products in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 86:2540‐2544.
   Butt, T.R., Edavettal, S.C., Hall, J.P., and Mattern, M.R. 2005. SUMO fusion technology for difficult‐to‐express proteins. Protein Expr. Purif. 43: 1‐9.
   Calderone, T.L., Stevens, R.D., and Oas, T.G. 1996. High‐level misincorporation of lysine for arginine at AGA codons in a fusion protein expressed in Escherichia coli. J. Mol. Biol. 262:407‐412.
   Canaves, J.M., Page, R., Wilson, I.A., and Stevens, R.C. 2004. Protein biophysical properties that correlate with crystallization success in Thermotoga maritima: Maximum clustering strategy for structural genomics. J. Mol. Biol. 344:977‐991.
   Carpousis, A.J. 2007. The RNA degradosome of Escherichia coli: An mRNA‐degrading machine assembled on RNase E. Annu. Rev. Microbiol. 61:71‐87.
   Cebe, R. and Geiser, M. 2006. Rapid and easy thermodynamic optimization of the 5′‐end of mRNA dramatically increases the level of wild type protein expression in Escherichia coli. Protein Expr. Purif. 45:374‐380.
   Chang, C.N., Kuang, W.J., and Chen, E.Y. 1986. Nucleotide‐sequence of the alkaline‐phosphatase gene of Escherichia coli. Gene 44:121‐125.
   Chang, J.Y. 1985. Thrombin specificity ‐ requirement for apolar amino‐acids adjacent to the thrombin cleavage site of polypeptide substrate. Eur. J. Biochem. 151:217‐224.
   Chen, L.F., Maloney, K., Krol, E., Zhu, B., and Yang, J. 2009. Cloning, overexpression, purification, and characterization of the maleylacetate reductase from sphingobium chlorophenolicum strain ATCC 53874. Curr. Microbiol. 58:599‐603.
   Chen, X., Tong, X.T., Xie, Y.H., Wang, Y., Ma, J.B., Gao, D.M., Wu, H.M., and Chen, H.B. 2006. Over‐expression and purification of isotopically labeled recombinant ligand‐binding domain of orphan nuclear receptor human B1‐binding factor/human liver receptor homologue 1 for NMR studies. Protein Expr. Purif. 45:99‐106.
   Chen, Y. and Leong, S.S.J. 2009. Adsorptive refolding of a highly disulfide‐bonded inclusion body protein using anion‐exchange chromatography. J. Chromatogr. A 1216:4877‐4886.
   Chen, Y., Song, J.M., Sui, S.F., and Wang, D.N. 2003. DnaK and DnaJ facilitated the folding process and reduced inclusion body formation of magnesium transporter CorA overexpressed in Escherichia coli. Protein Expr. Purif. 32:221‐231.
   Choi, S.I., Song, H.W., Moon, J.W., and Seong, B.L. 2001. Recombinant enterokinase light chain with affinity tag: Expression from Saccharomyces cerevisiae and its utilities in fusion protein technology. Biotechnol. Bioeng. 75:718‐724.
   Chong, S.R., Montello, G.E., Zhang, A.H., Cantor, E.J., Liao, W., Xu, M.Q., and Benner, J. 1998. Utilizing the C‐terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step. Nucleic Acids Res. 26:5109‐5115.
   Chou, C.P. 2007. Engineering cell physiology to enhance recombinant protein production in Escherichia coli. Appl. Microbiol. Biotechnol. 76:521‐532.
   Cinquin, O., Christopherson, R.I., and Menz, R.I. 2001. A hybrid plasmid for expression of toxic malarial proteins in Escherichia coli. Mol. Biochem. Parasitol. 117:245‐247.
   Collinsracie, L.A., McColgan, J.M., Grant, K.L., Diblasio‐Smith, E.A., McCoy, J.M., and Lavallie, E.R. 1995. Production of recombinant bovine enterokinase catalytic subunit in Escherichia coli using the novel secretory fusion partner Dsba. Biotechnology 13:982‐987.
   Couprie, J., Vinci, F., Dugave, C., Quemeneur, E., and Moutiez, M. 2000. Investigation of the DsbA mechanism through the synthesis and analysis of an irreversible enzyme‐ligand complex. Biochemistry 39:6732‐6742.
   Critton, D.A., Tortajada, A., Stetson, G., Peti, W., and Page, R. 2008. Structural basis of substrate recognition by hematopoietic tyrosine phosphatase. Biochemistry 47:13336‐13345.
   Cruz‐Vera, L.R., Magos‐Castro, M.A., Zamora‐Romo, E., and Guarneros, G. 2004. Ribosome stalling and peptidyl‐tRNA drop‐off during translational delay at AGA codons. Nucleic Acids Res. 32:4462‐4468.
   Dancheck, B., Nairn, A.C., and Peti, W. 2008. Detailed structural characterization of unbound protein phosphatase 1 inhibitors. Biochemistry 47:12346‐12356.
   Davis, G.D., Elisee, C., Newham, D.M., and Harrison, R.G. 1999. New fusion protein systems designed to give soluble expression in Escherichia coli. Biotechnol. Bioeng. 65:382‐388.
   de Marco, A. 2006. Two‐step metal affinity purification of double‐tagged (NusA‐His(6)) fusion proteins. Nat. Protoc. 1:1538‐1543.
   de Marco, A. 2009. Strategies for successful recombinant expression of disulfide bond‐dependent proteins in Escherichia coli. Microb. Cell Fact. 8:26.
   De Marco, V., Stier, G., Blandin, S., and de Marco, A. 2004. The solubility and stability of recombinant proteins are increased by their fusion to NusA. Biochem. Biophys. Res. Commun. 322:766‐771.
   Deboer, H.A., Comstock, L.J., and Vasser, M. 1983. The Tac promoter ‐ a functional hybrid derived from the Trp and Lac promoters. Proc. Natl. Acad. Sci. U.S.A. 80:21‐25.
   Delatorre, J.C., Ortin, J., Domingo, E., Delamarter, J., Allet, B., Davies, J., Bertrand, K.P., Wray, L.V., and Reznikoff, W.S. 1984. Plasmid vectors based on Tn10 DNA ‐ gene‐expression regulated by tetracycline. Plasmid 12:103‐110.
   DePristo, M.A., Zilversmit, M.M., and Hartl, D.L. 2006. On the abundance, amino acid composition, and evolutionary dynamics of low‐complexity regions in proteins. Gene 378:19‐30.
   Diguan, C., Li, P., Riggs, P.D., and Inouye, H. 1988. Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose‐binding protein. Gene 67:21‐30.
   Douette, P., Navet, R., Gerkens, P., Galleni, M., Levy, D., and Sluse, F.E. 2005. Escherichia coli fusion carrier proteins act as solubilizing agents for recombinant uncoupling protein 1 through interactions with GroEL. Biochem. Biophys. Res. Commun. 333:686‐693.
   Dvir, H. and Choe, S. 2009. Bacterial expression of a eukaryotic membrane protein in fusion to various Mistic orthologs. Protein Expr. Purif. 68:28‐33.
   Dyson, H.J. and Wright, P.E. 2005. Intrinsically unstructured proteins and their functions. Nat. Rev. Mol. Cell Biol. 6:197‐208.
   Dyson, M.R., Shadbolt, S.P., Vincent, K.J., Perera, R.L., and McCafferty, J. 2004. Production of soluble mammalian proteins in Escherichia coli: Identification of protein features that correlate with successful expression. BMC Biotechnol. 4:32.
   Elvin, C.M., Thompson, P.R., Argall, M.E., Hendry, P., Stamford, N.P.J., Lilley, P.E., and Dixon, N.E. 1990. Modified bacteriophage‐lambda promoter vectors for overproduction of proteins in Escherichia coli. Gene 87:123‐126.
   Esposito, D. and Chatterjee, D.K. 2006. Enhancement of soluble protein expression through the use of fusion tags. Curr. Opin. Biotechnol. 17:353‐358.
   Ferrer, M., Chernikova, T.N., Timmis, K.N., and Golyshin, P.N. 2004. Expression of a temperature‐sensitive esterase in a novel chaperone‐based Escherichia coli strain. Appl. Environ. Microbiol. 70:4499‐4504.
   Fox, J.D., Kapust, R.B., and Waugh, D.S. 2001. Single amino acid substitutions on the surface of Escherichia coli maltose‐binding protein can have a profound impact on the solubility of fusion proteins. Protein Sci. 10:622‐630.
   Gerdes, K., Christensen, S.K., and Lobner‐Olesen, A. 2005. Prokaryotic toxin‐antitoxin stress response loci. Nat. Rev. Microbiol. 3:371‐382.
   Goh, C.S., Lan, N., Douglas, S.M., Wu, B., Echols, N., Smith, A., Milburn, D., Montelione, G.T., Zhao, H., and Gerstein, M. 2004. Mining the structural genomics pipeline: Identification of protein properties that affect high‐throughput experimental analysis. J. Mol. Biol. 336:115‐130.
   Goldstein, J., Pollitt, N.S., and Inouye, M. 1990. Major cold shock protein of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 87:283‐287.
   Gordon, E., Horsefield, R., Swarts, H.G., de Pont, J.J., Neutze, R., and Snijder, A. 2008. Effective high‐throughput overproduction of membrane proteins in Escherichia coli. Protein Expr. Purif. 62:1‐8.
   Gottesman, S. 1990. Minimizing proteolysis in Escherichia coli ‐ Genetic solutions. Methods in Enzymol. 185:119‐129.
   Goulding, C.W. and Perry, L.J. 2003. Protein production in Escherichia coli for structural studies by X‐ray crystallography. J. Struct. Biol. 142:133‐143.
   Gråslund, S., Nordlund, P., Weigelt, J., Hallberg, B.M., Bray, J., Gileadi, O., Knapp, S., Oppermann, U., Arrowsmith, C., Hui, R., Ming, J., dhe‐Paganon, S., Park, H.W., Savchenko, A., Yee, A., Edwards, A., Vincentelli, R., Cambillau, C., Kim, R., Kim, S.H., Rao, Z., Shi, Y., Terwilliger, T.C., Kim, C.Y., Hung, L.W., Waldo, G.S., Peleg, Y., Albeck, S., Unger, T., Dym, O., Prilusky, J., Sussman, J.L., Stevens, R.C., Lesley, S.A., Wilson, I.A., Joachimiak, A., Collart, F., Dementieva, I., Donnelly, M.I., Eschenfeldt, W.H., Kim, Y., Stols, L., Wu, R., Zhou, M., Burley, S.K., Emtage, J.S., Sauder, J.M., Thompson, D., Bain, K., Luz, J., Gheyi, T., Zhang, F., Atwell, S., Almo, S.C., Bonanno, J.B., Fiser, A., Swaminathan, S., Studier, F.W., Chance, M.R., Sali, A., Acton, T.B., Xiao, R., Zhao, L., Ma, L.C., Hunt, J.F., Tong, L., Cunningham, K., Inouye, M., Anderson, S., Janjua, H., Shastry, R., Ho, C.K., Wang, D., Wang, H., Jiang, M., Montelione, G.T., Stuart, D.I., Owens, R.J., Daenke, S., Schutz, A., Heinemann, U., Yokoyama, S., Bussow, K., and Gunsalus, K.C. 2008a. Protein production and purification. Nat. Methods 5:135‐146.
   Gråslund, S., Sagemark, J., Berglund, H., Dahlgren, L.G., Flores, A., Hammarstroem, M., Johansson, I., Kotenyova, T., Nilsson, M., Nordlund, P., and Weigelt, J. 2008b. The use of systematic N‐ and C‐terminal deletions to promote production and structural studies of recombinant proteins. Protein Expr. Purif 58:210‐221.
   Grodberg, J. and Dunn, J.J. 1988. Ompt encodes the Escherichia coli outer‐membrane protease that cleaves T7‐Rna polymerase during purification. J. Bacteriol. 170:1245‐1253.
   Grunberg‐Manago, M. 1999. Messenger RNA stability and its role in control of gene expression in bacteria and phages. Ann. Rev. Genet. 33:193‐227.
   Guzman, L.M., Belin, D., Carson, M.J., and Beckwith, J. 1995. Tight regulation, modulation, and high‐level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177:4121‐4130.
   Hammarstrom, M., Hellgren, N., van Den Berg, S., Berglund, H., and Hard, T. 2002. Rapid screening for improved solubility of small human proteins produced as fusion proteins in Escherichia coli. Protein Sci. 11:313‐321.
   Harley, C.B. and Reynolds, R.P. 1987. Analysis of Escherichia coli promoter sequences. Nucleic Acids Res. 15:2343‐2361.
   Hartl, F.U. and Hayer‐Hartl, M. 2002. Protein folding ‐ Molecular chaperones in the cytosol: From nascent chain to folded protein. Science 295:1852‐1858.
   Hatfield, G.W. and Roth, D.A. 2007. Optimizing scaleup yield for protein production: Computationally Optimized DNA Assembly (CODA) and translation engineering. Biotechnol. Annu. Rev. 13:27‐42.
   Hawley, D.K. and McClure, W.R. 1983. Compilation and analysis of Escherichia coli promoter DNA‐sequences. Nucleic Acids Res. 11:2237‐2255.
   Huang, B.H., Shi, Z.T., and Tsai, M.D. 1994. A small, high‐copy‐number vector suitable for both in‐vitro and in‐vivo gene‐expression. Gene 151:143‐145.
   Hunke, S. and Betton, J.M. 2003. Temperature effect on inclusion body formation and stress response in the periplasm of Escherichia coli. Mol. Microbiol. 50:1579‐1589.
   Jaroszewski, L., Rychlewski, L., Li, Z., Li, W., and Godzik, A. 2005. FFAS03: A server for profile–profile sequence alignments. Nucleic Acids Res. 33:W284‐W288.
   Jenny, R.J., Mann, K.G., and Lundblad, R.L. 2003. A critical review of the methods for cleavage of fusion proteins with thrombin and factor Xa. Protein Expr. Purif. 31:1‐11.
   Jensen, P.Y., Bonander, N., Horn, N., Tumer, Z., and Farver, O. 1999. Expression, purification and copper‐binding studies of the first metal‐binding domain of Menkes protein. Eur. J. Biochem. 264:890‐896.
   Jing, G.Z., Huang, Z., Liu, Z.G., and Zou, Q. 1993. Plasmid Pkkh ‐ an improved vector with higher copy number for expression of foreign genes in Escherichia coli. Biotechnol. Lett. 15:439‐442.
   Johnston, K. and Marmorstein, R. 2003. Co‐expression of proteins in E. coli using dual expression vectors. Methods Mol. Biol. 205:205‐213.
   Jones, D.T. 1999. Protein secondary structure prediction based on position‐specific scoring matrices. J. Mol. Biol. 292:195‐202.
   Jones, P.G., Vanbogelen, R.A., and Neidhardt, F.C. 1987. Induction of proteins in response to low‐temperature in Escherichia coli. J. Bacteriol. 169:2092‐2095.
   Joseph, R.E. and Andreotti, A.H. 2008. Bacterial expression and purification of Interleukin‐2 Tyrosine kinase: Single step separation of the chaperonin impurity. Protein Expr. Purif. 60:194‐197.
   Kane, J.F. 1995. Effects of rare codon clusters on high‐level expression of heterologous proteins in Escherichia coli. Curr. Opin. Biotechnol. 6:494‐500.
   Kaplan, W., Husler, P., Klump, H., Erhardt, J., Sluis Cremer, N., and Dirr, H. 1997. Conformational stability of pGEX‐expressed Schistosoma japonicum glutathione S‐transferase: A detoxification enzyme and fusion‐protein affinity tag. Protein Sci. 6:399‐406.
   Kapust, R.B. and Waugh, D.S. 1999. Escherichia coli maltose‐binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci. 8:1668‐1674.
   Kapust, R.B. and Waugh, D.S. 2000. Controlled intracellular processing of fusion proteins by TEV protease. Protein Expr. Purif. 19:312‐318.
   Kapust, R.B., Tozser, J., Fox, J.D., Anderson, D.E., Cherry, S., Copeland, T.D., and Waugh, D.S. 2001. Tobacco etch virus protease: Mechanism of autolysis and rational design of stable mutants with wild‐type catalytic proficiency. Protein Eng. 14:993‐1000.
   Kapust, R.B., Tozser, J., Copeland, T.D., and Waugh, D.S. 2002. The P1 ′ specificity of tobacco etch virus protease. Biochem. Biophys. Res. Commun. 294:949‐955.
   Karlin, S., Brocchieri, L., Bergman, A., Mrazek, J., and Gentles, A.J. 2002. Amino acid runs in eukaryotic proteomes and disease associations. Proc. Natl. Acad. Sci. U.S.A. 99:333‐338.
   Kataeva, I., Chang, J., Xu, H., Luan, C.H., Zhou, J., Uversky, V.N., Lin, D., Horanyi, P., Liu, Z.J., Ljungdahl, L.G., Rose, J., Luo, M., and Wang, B.C. 2005. Improving solubility of Shewanella oneidensis MR‐1 and Clostridium thermocellum JW‐20 proteins expressed into Esherichia coli. J. Proteome Res. 4:1942‐1951.
   Kelker, M.S., Page, R., and Peti, W. 2009. Crystal structures of protein phosphatase‐1 bound to nodularin‐R and tautomycin: A novel scaffold for structure‐based drug design of serine/threonine phosphatase inhibitors. J. Mol. Biol. 385:11‐21.
   Kikuchi, Y., Yoda, K., Yamasaki, M., and Tamura, G. 1981. The nucleotide‐sequence of the promoter and the amino‐terminal region of alkaline‐phosphatase structural gene (Phoa) of Escherichia coli. Nucleic Acids Res. 9:5671‐5678.
   Kishore, U., Leigh, L.E.A., Eggleton, P., Strong, P., Perdikoulis, M.V., Willis, A.C., and Reid, K.B.M. 1998. Functional characterization of a recombinant form of the C‐terminal, globular head region of the B‐chain of human serum complement protein, C1q. Biochem. J. 333:27‐32.
   Klock, H.E., Koesema, E.J., Knuth, M.W., and Lesley, S.A. 2008. Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts. Proteins 71:982‐994.
   Kohl, T., Schmidt, C., Wiemann, S., Poustka, A., and Korf, U. 2008. Automated production of recombinant human proteins as resource for proteome research. Proteome Sci. 6:10.
   Kyratsous, C.A., Silverstein, S.J., DeLong, C.R., and Panagiotidis, C.A. 2009. Chaperone‐fusion expression plasmid vectors for improved solubility of recombinant proteins in Escherichia coli. Gene 440:9‐15.
   Langlais, C., Guilleaume, B., Wermke, N., Scheuermann, T., Ebert, L., LaBaer, J., and Korn, B. 2007. A systematic approach for testing expression of human full‐length proteins in cell‐free expression systems. BMC Biotechnol. 7:64.
   Lauber, T., Marx, U.C., Schulz, A., Kreutzmann, P., Rosch, P., and Hoffmann, S. 2001. Accurate disulfide formation in Escherichia coli: Overexpression and characterization of the first domain (HF6478) of the multiple Kazal‐type inhibitor LEKTI. Protein Expr. Purif. 22:108‐112.
   LaVallie, E.R., DiBlasio, E.A., Kovacic, S., Grant, K.L., Schendel, P.F., and McCoy, J.M. 1993. A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Biotechnology 11:187‐193.
   Lee, C.D., Sun, H.C., Hu, S.M., Chiu, C.F., Homhuan, A., Liang, S.M., Leng, C.H., and Wang, T.F. 2008. An improved SUMO fusion protein system for effective production of native proteins. Protein Sci. 17:1241‐1248.
   Lee, H.S., Berger, D.K., and Kustu, S. 1993. Activity of purified nifa, a transcriptional activator of nitrogen‐fixation genes. Proc. Natl. Acad. Sci. U.S.A. 90:2266‐2270.
   Lee, N., Francklyn, C., and Hamilton, E.P. 1987. Arabinose‐induced binding of arac protein to aral2 activates the arabad operon promoter. Proc. Natl. Acad. Sci. U.S.A. 84:8814‐8818.
   Lefebvre, J., Boileau, G., and Manjunath, P. 2009a. Recombinant expression and affinity purification of a novel epididymal human sperm‐binding protein, BSPH1. Mol. Hum. Reprod. 15:105‐114.
   Lefebvre, J., Boileau, G., and Manjunath, P. 2009b. Recombinant expression and affinity purification of a novel epididymal human sperm‐binding protein, BSPH1. Mol. Hum. Reprod. 15:105‐114.
   Leichert, L.I. and Jakob, U. 2004. Protein thiol modifications visualized in vivo. PLoS Biol. 2:e333.
   Lopez, P.J., Marchand, I., Joyce, S.A., and Dreyfus, M. 1999. The C‐terminal half of RNase E, which organizes the Escherichia coli degradosome, participates in mRNA degradation but not rRNA processing in vivo. Mol. Microbiol. 33:188‐199.
   Lorimer, G.H. 1996. A quantitative assessment of the role of the chaperonin proteins in protein folding in vivo. FASEB J. 10:5‐9.
   Malakhov, M.P., Mattern, M.R., Malakhova, O.A., Drinker, M., Weeks, S.D., and Butt, T.R. 2004. SUMO fusions and SUMO‐specific protease for efficient expression and purification of proteins. J. Struct. Funct. Genomics 5:75‐86.
   Malik, A., Jenzsch, M., Lubbert, A., Rudolph, R., and Sohling, B. 2007. Periplasmic production of native human proinsulin as a fusion to E. coli ecotin. Protein Expr. Purif. 55:100‐111.
   Milisavljevic, M.D., Papic, D.R., Timotijevic, G.S., and Maksimovic, V.R. 2009. Successful production of recombinant buckwheat cysteine‐rich aspartic protease in Escherichia coli. J. Serb. Chem. Soc. 74:607‐618.
   Miyada, C.G., Stoltzfus, L., and Wilcox, G. 1984. Regulation of the arac gene of Escherichia coli ‐ Catabolite repression, auto‐regulation, and effect on arabad expression. Proc. Natl. Acad. Sci. U.S.A. 81:4120‐4124.
   Miyake, T., Oka, T., Nishizawa, T., Misoka, F., Fuwa, T., Yoda, K., Yamasaki, M., and Tamura, G. 1985. Secretion of human interferon‐alpha induced by using secretion vectors containing a promoter and signal sequence of alkaline‐phosphatase gene of Escherichia coli. J. Biochem. 97:1429‐1436.
   Miyashita, K., Kusumi, M., Utsumi, R., Komano, T., and Satoh, N. 1992. Expression and purification of recombinant 3c‐proteinase of coxsackievirus‐B3. Biosci. Biotechnol. Biochem. 56:746‐750.
   Mobley, C.K., Myers, J.K., Hadziselimovic, A., Ellis, C.D., and Sanders, C.R. 2007. Purification and initiation of structural characterization of human peripheral myelin protein 22, an integral membrane protein linked to peripheral neuropathies. Biochemistry 46:11185‐11195.
   Moffatt, B.A. and Studier, F.W. 1987. T7 lysozyme inhibits transcription by T7 rna‐polymerase. Cell 49:221‐227.
   Mohanty, A.K. and Wiener, M.C. 2004. Membrane protein expression and production: Effects of polyhistidine tag length and position. Protein Expr. Purif. 33:311‐325.
   Mohanty, A.K., Simmons, C.R., and Wiener, M.C. 2003. Inhibition of tobacco etch virus protease activity by detergents. Protein Expr. Purif. 27:109‐114.
   Mustelin, T., Tautz, L., and Page, R. 2005. Structure of the hematopoietic tyrosine phosphatase (HePTP) catalytic domain: Structure of a KIM phosphatase with phosphate bound at the active site. J. Mol. Biol. 354:150‐163.
   Nagai, K. and Thogersen, H.C. 1984. Generation of beta‐globin by sequence‐specific proteolysis of a hybrid protein produced in Escherichia coli. Nature 309:810‐812.
   Nallamsetty, S. and Waugh, D.S. 2006. Solubility‐enhancing proteins MBP and NusA play a passive role in the folding of their fusion partners. Protein Expr. Purif. 45:175‐182.
   Nallamsetty, S. and Waugh, D.S. 2007. Mutations that alter the equilibrium between open and closed conformations of Escherichia coli maltose‐binding protein impede its ability to enhance the solubility of passenger proteins. Biochem. Biophys. Res. Commun. 364:639‐644.
   Netzer, W.J. and Hartl, F.U. 1997. Recombination of protein domains facilitated by co‐translational folding in eukaryotes. Nature 388:343‐349.
   Niiranen, L., Espelid, S., Karlsen, C.R., Mustonen, M., Paulsen, S.M., Heikinheimo, P., and Willassen, N.P. 2007. Comparative expression study to increase the solubility of cold adapted Vibrio proteins in Escherichia coli. Protein Expr. Purif. 52:210‐218.
   Nilsson, B., Moks, T., Jansson, B., Abrahmsen, L., Elmblad, A., Holmgren, E., Henrichson, C., Jones, T.A., and Uhlen, M. 1987. A synthetic igg‐binding domain based on staphylococcal protein‐A. Protein Eng. 1:107‐113.
   Nilsson, J., Larsson, M., Stahl, S., Nygren, P.A., and Uhlen, M. 1996. Multiple affinity domains for the detection, purification and immobilization of recombinant proteins. J. Mol. Recognit. 9:585‐594.
   Oberg, K., Chrunyk, B.A., Wetzel, R., and Fink, A.L. 1994. Native‐like secondary structure in interleukin‐1‐beta inclusion‐bodies by attenuated total reflectance Ftir. Biochemistry 33:2628‐2634.
   Ohana, R.F., Enccell, L.P., Zhao, K., Simpson, D., Slater, M.R., Urh, M., and Wood, K.V. 2009. HaloTag7: A genetically engineered tag that enhances bacterial expression of soluble proteins and improves protein purification. Protein Expr. Purif. 68:110‐120.
   Olins, P.O. and Rangwala, S.H. 1990. Vector for enhanced translation of foreign genes in Escherichia coli. Methods Enzymol. 185:115‐119.
   Otto, C.M., Niagro, F., Su, X.Z., and Rawlings, C.A. 1995. Expression of recombinant feline tumor‐necrosis‐factor is toxic to Escherichia coli. Clin. Diagn. Lab. Immunol. 2:740‐746.
   Park, S.L., Kwon, M.J., Kim, S.K., and Nam, S.W. 2004. GroEL/ES chaperone and low culture temperature synergistically enhanced the soluble expression of CGTase in E‐coli. J. Microbiol. Biotechnol. 14:216‐219.
   Pedersen, K., Zavialov, A.V., Pavlov, M.Y., Elf, J., Gerdes, K., and Ehrenberg, M. 2003. The bacterial toxin RelE displays codon‐specific cleavage of mRNAs in the ribosomal A site. Cell 112:131‐140.
   Peng, L., Xu, Z.N., Fang, X.M., Wang, F., Yang, S., and Cen, P.L. 2004. Preferential codons enhancing the expression level of human beta‐defensin‐2 in recombinant Escherichia coli. Protein Pept. Lett. 11:339‐344.
   Peti, W. and Page, R. 2007. Strategies to maximize heterologous protein expression in Escherichia coli with minimal cost. Protein Expr. Purif. 51:1‐10.
   Phan, J., Zdanov, A., Evdokimov, A.G., Tropea, J.E., Peters, H.K., Kapust, R.B., Li, M., Wlodawer, A., and Waugh, D.S. 2002. Structural basis for the substrate specificity of tobacco etch virus protease. J. Biol. Chem. 277:50564‐50572.
   Phillips, T.A., Vanbogelen, R.A., and Neidhardt, F.C. 1984. Ion gene‐product of Escherichia coli is a heat‐shock protein. J. Bacteriol. 159:283‐287.
   Pinsach, J., de Mas, C., Lopez‐Santin, J., Striedner, G., and Bayer, K. 2008. Influence of process temperature on recombinant enzyme activity in Escherichia coli fed‐batch Cultures. Enzyme Microb. Technol. 43:507‐512.
   Piserchio, A., Ghose, R., and Cowburn, D. 2009. Optimized bacterial expression and purification of the c‐Src catalytic domain for solution NMR studies. J. Biomol. NMR 44:87‐93.
   Porath, J., Carlsson, J., Olsson, I., and Belfrage, G. 1975. Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258:598‐599.
   Prinz, W.A., Aslund, F., Holmgren, A., and Beckwith, J. 1997. The role of the thioredoxin and glutaredoxin pathways in reducing protein disulfide bonds in the Escherichia coli cytoplasm. J. Biol. Chem. 272:15661‐15667.
   Pryor, K.D. and Leiting, B. 1997. High‐level expression of soluble protein in Escherichia coli using a His(6)‐tag and maltose‐binding‐protein double‐affinity fusion system. Protein Expr. Purif. 10:309‐319.
   Qing, G., Ma, L.C., Khorchid, A., Swapna, G.V., Mal, T.K., Takayama, M.M., Xia, B., Phadtare, S., Ke, H., Acton, T., Montelione, G.T., Ikura, M., and Inouye, M. 2004. Cold‐shock induced high‐yield protein production in Escherichia coli. Nat. Biotechnol. 22:877‐882.
   Reilly, D. and Fairbrother, W.J. 1994. A novel isotope labeling protocol for bacterially expressed Proteins. J. Biomol. NMR 4:459‐462.
   Riggs, P. 2000. Expression and purification of recombinant proteins by fusion to maltose‐binding protein. Mol. Biotechnol. 15:51‐63.
   Ritz, D. and Beckwith, J. 2001. Roles of thiol‐redox pathways in bacteria. Annu. Rev. Microbiol. 55:21‐48.
   Rosenberg, M. and Court, D. 1979. Regulatory sequences involved in the promotion and termination of rna‐transcription. Annu. Rev. Genet. 13:319‐353.
   Routzahn, K. and Waugh, D. 2002. Differential effects of supplementary affinity tags on the solubility of MBP fusion proteins. J. Struct. Funct. Genomics 2:83‐92.
   Saavedraalanis, V.M., Rysavy, P., Rosenberg, L.E., and Kalousek, F. 1994. Rat‐liver mitochondrial processing peptidase ‐ both alpha‐subunit and beta‐subunit are required for activity. J. Biol. Chem. 269:9284‐9288.
   Sahdev, S., Khattar, S.K., and Saini, K.S. 2008. Production of active eukaryotic proteins through bacterial expression systems: A review of the existing biotechnology strategies. Mol. Cell Biochem. 307:249‐264.
   Sahu, S.K., Rajasekharan, A., and Gummadi, S.N. 2009. GroES and GroEL are essential chaperones for refolding of recombinant human phospholipid scramblase 1 in E. coli. Biotechnol Lett. 31:1745‐1752.
   Sakharkar, M.K., Kangueane, P., Sakharkar, K.R., and Zhong, Z. 2006. Huge proteins in the human proteome and their participation in hereditary diseases. In Silico Biol. 6:275‐279.
   Sati, S.P., Singh, S.K., Kumar, N., and Sharma, A. 2002. Extra terminal residues have a profound effect on the folding and solubility of a Plasmodium falciparum sexual stage‐specific protein over‐expressed in Escherichia coli. Eur. J. Biochem. 269:5259‐5263.
   Schenk, P.M., Baumann, S., Mattes, R., and Steinbiss, H.H. 1995. Improved high‐level expression system for eukaryotic genes in Escherichia coli using T7 RNA polymerase and rare ArgtRNAs. Biotechniques 19:196‐200.
   Seeliger, M.A., Young, M., Henderson, M.N., Pellicena, P., King, D.S., Falick, A.M., and Kuriyan, J. 2005. High yield bacterial expression of active c‐Abl and c‐Src tyrosine kinases. Protein Sci. 14:3135‐3139.
   Sharp, P.M. and Li, W.H. 1987. The codon Adaptation Index–a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res. 15:1281‐1295.
   Shirakawa, M., Tsurimoto, T., and Matsubara, K. 1984. Plasmid vectors designed for high‐efficiency expression controlled by the portable reca promoter‐operator of Escherichia coli. Gene 28:127‐132.
   Shirano, Y. and Shibata, D. 1990. Low temperature cultivation of Escherichia coli carrying a rice lipoxygenase L‐2 cDNA produces a soluble and active enzyme at a high level. FEBS Lett. 271:128‐130.
   Singleton, S.F., Simonette, R.A., Sharma, N.C., and Roca, A.I. 2002. Intein‐mediated affinity‐fusion purification of the Escherichia coli RecA protein. Protein Expr. Purif. 26:476‐488.
   Skerra, A. 1994. Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli. Gene 151:131‐135.
   Smith, D.B. and Johnson, K.S. 1988. Single‐step purification of polypeptides expressed in Escherichia coli as fusions with glutathione s‐transferase. Gene 67:31‐40.
   Spiess, C., Beil, A., and Ehrmann, M. 1999. A temperature‐dependent switch from chaperone to protease in a widely conserved heat shock protein. Cell 97:339‐347.
   Spirin, A.S. 2004. High‐throughput cell‐free systems for synthesis of functionally active proteins. Trends Biotechnol. 22:538‐545.
   Stewart, E.J., Aslund, F., and Beckwith, J. 1998. Disulfide bond formation in the Escherichia coli cytoplasm: An in vivo role reversal for the thioredoxins. EMBO J. 17:5543‐5550.
   Stieber, D., Gabant, P., and Szpirer, C. 2008. The art of selective killing: Plasmid toxin/antitoxin systems and their technological applications. Biotechniques 45:344‐346.
   Studier, F.W. 1991. Use of bacteriophage‐T7 lysozyme to improve an inducible T7 expression system. J. Mol. Biol. 219:37‐44.
   Studier, F.W. and Moffatt, B.A. 1986a. Use of bacteriophage T7 RNA polymerase to direct selective high‐level expression of cloned genes. J. Mol. Biol. 189:113‐130.
   Studier, F.W. and Moffatt, B.A. 1986b. Use of bacteriophage‐T7 rna‐polymerase to direct selective high‐level expression of cloned genes. J. Mol. Biol. 189:113‐130.
   Studier, F.W., Rosenberg, A.H., Dunn, J.J., and Dubendorff, J.W. 1990. Use of T7 rna‐polymerase to direct expression of cloned genes. Methods Enzymol. 185:60‐89.
   Tanaka, T., Kubota, M., Samizo, K., Nakajima, Y., Hoshino, M., Kohno, T., and Wakamatsu, E. 1999. One‐step affinity purification of the G protein beta gamma subunits from bovine brain using a histidine‐tagged G protein alpha subunit. Protein Expr. Purif. 15:207‐212.
   Tegel, H., Steen, J., Konrad, A., Nikdin, H., Pettersson, K., Stenvall, M., Tourle, S., Wrethagen, U., Xu, L., Yderland, L., Uhlen, M., Hober, S., and Ottosson, J. 2009. High‐throughput protein production–lessons from scaling up from 10 to 288 recombinant proteins per week. Biotechnol. J. 4:51‐57.
   Terpe, K. 2003. Overview of tag protein fusions: From molecular and biochemical fundamentals to commercial systems. Appl. Microbiol. Biotechnol. 60:523‐533.
   Tsumoto, K., Ejima, D., Kumagai, I., and Arakawa, T. 2003. Practical considerations in refolding proteins from inclusion bodies. Protein Expr. Purif. 28:1‐8.
   Turner, P., Holst, O., and Karlsson, E.N. 2005. Optimized expression of soluble cyclomaltodextrinase of thermophilic origin in Escherichia coli by using a soluble fusion‐tag and by tuning of inducer concentration. Protein Expr. Purif. 39:54‐60.
   Valax, P. and Georgiou, G. 1993. Molecular characterization of beta‐lactamase inclusion‐bodies produced in Escherichia coli. 1. Composition. Biotechnol. Prog. 9:539‐547.
   Vasina, J.A. and Baneyx, F. 1996. Recombinant protein expression at low temperatures under the transcriptional control of the major Escherichia coli cold shock promoter cspA. Appl. Environ. Microbiol. 62:1444‐1447.
   Vasina, J.A. and Baneyx, F. 1997. Expression of aggregation‐prone recombinant proteins at low temperatures: A comparative study of the Escherichia coli cspA and tac promoter systems. Protein Expr. Purif. 9:211‐218.
   Veldkamp, C.T., Peterson, F.C., Hayes, P.L., Mattmiller, J.E., Haugner, J.C., de la Cruz, N., and Volkman, B.F. 2007. On‐column refolding of recombinant chemokines for NMR studies and biological assays. Protein Expr. Purif. 52:202‐209.
   Vincentelli, R., Bignon, C., Gruez, A., Canaan, S., Sulzenbacher, G., Tegoni, M., Campanacci, V., and Cambillau, C. 2003. Medium‐scale structural genomics: Strategies for protein expression and crystallization. Accounts Chem. Res. 36:165‐172.
   Volonte, F., Marinelli, F., Gastaldo, L., Sacchi, S., Pilone, M.S., Pollegioni, L., and Molla, G. 2008. Optimization of glutaryl‐7‐aminocephalosporanic acid acylase expression in E‐coli. Protein Expr. Purif. 61:131‐137.
   Wakagi, T., Oshima, T., Imamura, H., and Matsuzawa, H. 1998. Cloning of the gene for inorganic pyrophosphatase from a thermoacidophilic archaeon, Sulfolobus sp. strain 7, and overproduction of the enzyme by coexpression of tRNA for arginine rare codon. Biosci. Biotechnol. Biochem. 62:2408‐2414.
   Walsh, G. 2006. Biopharmaceutical benchmarks 2006. Nature Biotechnol. 24:769‐765.
   Wang, W.R., Marimuthu, A., Tsai, J., Kumar, A., Krupka, H.I., Zhang, C., Powell, B., Suzuki, Y., Nguyen, H., Tabrizizad, M., Luu, C., and West, B.L. 2006. Structural characterization of autoinhibited c‐Met kinase produced by coexpression in bacteria with phosphatase. Proc. Natl. Acad. Sci. U.S.A. 103:3563‐3568.
   Wang, Y.H., Ayrapetov, M.K., Lin, X.F., and Sun, G.Q. 2006. A new strategy to produce active human Src from bacteria for biochemical study of its regulation. Biochem. Biophys. Res. Commun. 346:606‐611.
   Winter, J., Neubauer, P., Glockshuber, R., and Rudolph, R. 2000. Increased production of human proinsulin in the periplasmic space of Escherichia coli by fusion to DsbA. J. Biotechnol. 84:175‐185.
   Wittliff, J.L., Wenz, L.L., Dong, J., Nawaz, Z., and Butt, T.R. 1990. Expression and characterization of an active human estrogen‐receptor as a ubiquitin fusion protein from Escherichia coli. J. Biol. Chem. 265:22016‐22022.
   Xu, Y., Yasin, A., Tang, R., Scharer, J.M., Moo‐Young, M., and Chou, C.P. 2008. Heterologous expression of lipase in Escherichia coli is limited by folding and disulfide bond formation. Appl. Microbiol. Biotechnol. 81:79‐87.
   Yan, F., Qian, M.L., Yang, F., Cai, F., Yuan, Z., Lai, S.T., Zhao, X.Y., Gou, L.T., Hu, Z.G., and Deng, H.X. 2007. A novel pro‐apoptosis protein PNAS‐4 from Xenopus laevis: Cloning, expression, purification, and polyclonal antibody production. Biochemistry (Moscow) 72:664‐671.
   Yao, J.W., Patrone, J.D., and Dotson, G.D. 2009. Characterization and kinetics of phosphopantothenoylcysteine synthetase from Enterococcus faecalis. Biochemistry 48:2799‐2806.
   Yeo, Y.J., Shin, S., Lee, S.G., Park, S., and Jeong, Y.J. 2009. Production, purification, and characterization of soluble NADH‐flavin Oxidoreductase (StyB) from Pseudomonas putida SN1. J. Microbiol. Biotechnol. 19:362‐367.
   Yin, J.C., Li, G.X., Ren, X.F., and Herrler, G. 2007. Select what you need: A comparative evaluation of the advantages and limitations of frequently used expression systems for foreign genes. J. Biotechnol. 127:335‐347.
   Zhang, Y.B., Howitt, J., McCorkle, S., Lawrence, P., Springer, K., and Freimuth, P. 2004. Protein aggregation during overexpression limited by peptide extensions with large net negative charge. Protein Expr. Purif. 36:207‐216.
   Zhang, Z.W., Gildersleeve, J., Yang, Y.Y., Xu, R., Loo, J.A., Uryu, S., Wong, C.H., and Schultz, P.G. 2004. A new strategy for the synthesis of glycoproteins. Science 303:371‐373.
   Zhao, Y.X., Benita, Y., Lok, M., Kuipers, B., van der Ley, P., Jiskoot, W., Hennink, W.E., Crommelin, D.J.A., and Oosting, R.S. 2005. Multi‐antigen immunization using IgG binding domain ZZ as carrier. Vaccine 23:5082‐5090.
   Zuo, X., Li, S., Hall, J., Mattern, M.R., Tran, H., Shoo, J., Tan, R., Weiss, S.R., and Butt, T.R. 2005a. Enhanced expression and purification of membrane proteins by SUMO fusion in Escherichia coli. J. Struct. Funct. Genomics 6:103‐111.
   Zuo, X., Mattern, M.R., Tan, R., Li, S., Hall, J., Sterner, D.E., Shoo, J., Tran, H., Lim, P., Sarafianos, S.G., Kazi, L., Navas‐Martin, S., Weiss, S.R., and Butt, T.R. 2005b. Expression and purification of SARS coronavirus proteins using SUMO‐fusions. Protein Expr. Purif. 42:100‐110.
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