热门搜索词：
Strategies to Optimize Protein Expression in E. coli
关键词： strategies to optimize来源： 互联网
- Abstract
- Table of Contents
- Figures
- Literature Cited
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
GO TO THE FULL PROTOCOL: PDF or HTML at Wiley Online Library 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
GO TO THE FULL PROTOCOL: PDF or HTML at Wiley Online Library Materials
GO TO THE FULL PROTOCOL: PDF or HTML at Wiley Online Library Figures
-
View ImageFigure 5.24.1 Schematic overview of the topics covered in this review, highlighting the multiple parameters (listed on the right) that can greatly impact the success of soluble expression. -
View ImageFigure 5.24.2 Flowchart of a general expression protocol used by the authors to express a broad range of targets, from phosphatases, to neuronal scaffolding proteins, to bacterial signaling proteins. The approximate time required to complete each segment of the protocol is listed to the left of the corresponding step. -
View ImageFigure 5.24.3 Flowchart depicting the critical factors to consider, common obstacles, and potential solutions for each stage of protein expression in E. coli . The left column lists the major steps of recombinant protein expression with key variables to consider. The middle column includes common obstacles encountered at each step, while possible solutions for each obstacle are presented in the right column.
Videos
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 |
推荐方法
移动版：