This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Makrides, S. C. Search for Related Content PubMed PubMed Citation Articles by Makrides, S. C.

 Previous Article  |  Next Article 

Microbiol. Rev., Sep 1996, 512-538, Vol 60, No. 3
Copyright © 1996, American Society for Microbiology

Strategies for achieving high-level expression of genes in Escherichia coli

SC Makrides
Department of Molecular Biology, T Cell Sciences, Inc., Needham, Massachusetts 02194, USA.

Progress in our understanding of several biological processes promises to broaden the usefulness of Escherichia coli as a tool for gene expression. There is an expanding choice of tightly regulated prokaryotic promoters suitable for achieving high-level gene expression. New host strains facilitate the formation of disulfide bonds in the reducing environment of the cytoplasm and offer higher protein yields by minimizing proteolytic degradation. Insights into the process of protein translocation across the bacterial membranes may eventually make it possible to achieve robust secretion of specific proteins into the culture medium. Studies involving molecular chaperones have shown that in specific cases, chaperones can be very effective for improved protein folding, solubility, and membrane transport. Negative results derived from such studies are also instructive in formulating different strategies. The remarkable increase in the availability of fusion partners offers a wide range of tools for improved protein folding, solubility, protection from proteases, yield, and secretion into the culture medium, as well as for detection and purification of recombinant proteins. Codon usage is known to present a potential impediment to high-level gene expression in E. coli. Although we still do not understand all the rules governing this phenomenon, it is apparent that "rare" codons, depending on their frequency and context, can have an adverse effect on protein levels. Usually, this problem can be alleviated by modification of the relevant codons or by coexpression of the cognate tRNA genes. Finally, the elucidation of specific determinants of protein degradation, a plethora of protease-deficient host strains, and methods to stabilize proteins afford new strategies to minimize proteolytic susceptibility of recombinant proteins in E. coli.

This article has been cited by other articles:

• Smialowski, P., Martin-Galiano, A. J., Mikolajka, A., Girschick, T., Holak, T. A., Frishman, D. (2007). Protein solubility: sequence based prediction and experimental verification. Bioinformatics 23: 2536-2542 [Abstract] [Full Text]  
• Rabhi-Essafi, I., Sadok, A., Khalaf, N., Fathallah, D. M. (2007). A strategy for high-level expression of soluble and functional human interferon {alpha} as a GST-fusion protein in E.coli. Protein Eng Des Sel 20: 201-209 [Abstract] [Full Text]  
• Sletta, H., Tondervik, A., Hakvag, S., Aune, T. E. V., Nedal, A., Aune, R., Evensen, G., Valla, S., Ellingsen, T. E., Brautaset, T. (2007). The Presence of N-Terminal Secretion Signal Sequences Leads to Strong Stimulation of the Total Expression Levels of Three Tested Medically Important Proteins during High-Cell-Density Cultivations of Escherichia coli. Appl. Environ. Microbiol. 73: 906-912 [Abstract] [Full Text]  
• Seleem, M. N., Ali, M., Boyle, S. M., Mukhopadhyay, B., Witonsky, S. G., Schurig, G. G., Sriranganathan, N. (2006). Establishment of a Gene Expression System in Ochrobactrum anthropi.. Appl. Environ. Microbiol. 72: 6833-6836 [Abstract] [Full Text]  
• Lack, G., Homberger-Zizzari, E., Folkers, G., Scapozza, L., Perozzo, R. (2006). Recombinant Expression and Biochemical Characterization of the Unique Elongating beta-Ketoacyl-Acyl Carrier Protein Synthase Involved in Fatty Acid Biosynthesis of Plasmodium falciparum Using Natural and Artificial Substrates. J. Biol. Chem. 281: 9538-9546 [Abstract] [Full Text]  
• Fisher, A. C., Kim, W., Delisa, M. P. (2006). Genetic selection for protein solubility enabled by the folding quality control feature of the twin-arginine translocation pathway. Protein Sci. 15: 449-458 [Abstract] [Full Text]  
• Idicula-Thomas, S., Kulkarni, A. J., Kulkarni, B. D., Jayaraman, V. K., Balaji, P. V. (2006). A support vector machine-based method for predicting the propensity of a protein to be soluble or to form inclusion body on overexpression in Escherichia coli. Bioinformatics 22: 278-284 [Abstract] [Full Text]  
• Royo, J. L., Manyani, H., Cebolla, A., Santero, E. (2005). A new generation of vectors with increased induction ratios by overimposing a second regulatory level by attenuation. Nucleic Acids Res 33: e169-e169 [Abstract] [Full Text]  
• Xu, Y., Weng, C.-L., Narayanan, N., Hsieh, M.-Y., Anderson, W. A., Scharer, J. M., Moo-Young, M., Chou, C. P. (2005). Chaperone-Mediated Folding and Maturation of the Penicillin Acylase Precursor in the Cytoplasm of Escherichia coli. Appl. Environ. Microbiol. 71: 6247-6253 [Abstract] [Full Text]  
• Sato, Y., Monincova, M., Chaloupkova, R., Prokop, Z., Ohtsubo, Y., Minamisawa, K., Tsuda, M., Damborsky, J., Nagata, Y. (2005). Two Rhizobial Strains, Mesorhizobium loti MAFF303099 and Bradyrhizobium japonicum USDA110, Encode Haloalkane Dehalogenases with Novel Structures and Substrate Specificities. Appl. Environ. Microbiol. 71: 4372-4379 [Abstract] [Full Text]  
• Eom, G. T., Song, J. K., Ahn, J. H., Seo, Y. S., Rhee, J. S. (2005). Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System. Appl. Environ. Microbiol. 71: 3468-3474 [Abstract] [Full Text]  
• Kimura, S., Umemura, T., Iyanagi, T. (2005). Two-Cistronic Expression Plasmids for High-Level Gene Expression in Escherichia coli Preventing Translational Initiation Inhibition Caused by the Intramolecular Local Secondary Structure of mRNA. J Biochem 137: 523-533 [Abstract] [Full Text]  
• Idicula-Thomas, S., Balaji, P. V. (2005). Understanding the relationship between the primary structure of proteins and its propensity to be soluble on overexpression in Escherichia coli. Protein Sci. 14: 582-592 [Abstract] [Full Text]  
• Lee, S. Y., Yang, J.-S., Hong, W., Premont, R. T., Hsu, V. W. (2005). ARFGAP1 plays a central role in coupling COPI cargo sorting with vesicle formation. JCB 168: 281-290 [Abstract] [Full Text]  
• Richard, C., Drider, D., Elmorjani, K., Marion, D., Prevost, H. (2004). Heterologous Expression and Purification of Active Divercin V41, a Class IIa Bacteriocin Encoded by a Synthetic Gene in Escherichia coli. J. Bacteriol. 186: 4276-4284 [Abstract] [Full Text]  
• Choi, Y. J., Miguez, C. B., Lee, B. H. (2004). Characterization and Heterologous Gene Expression of a Novel Esterase from Lactobacillus casei CL96. Appl. Environ. Microbiol. 70: 3213-3221 [Abstract] [Full Text]  
• Paulus, M., Haslbeck, M., Watzele, M. (2004). RNA stem-loop enhanced expression of previously non-expressible genes. Nucleic Acids Res 32: e78-e78 [Abstract] [Full Text]  
• Belin, P., Dassa, J., Drevet, P., Lajeunesse, E., Savatier, A., Boulain, J.-C., Menez, A. (2004). Toxicity-based selection of Escherichia coli mutants for functional recombinant protein production: application to an antibody fragment. Protein Eng Des Sel 17: 491-500 [Abstract] [Full Text]  
• Fodor, B. D., Kovacs, A. T., Csaki, R., Hunyadi-Gulyas, E., Klement, E., Maroti, G., Meszaros, L. S., Medzihradszky, K. F., Rakhely, G., Kovacs, K. L. (2004). Modular Broad-Host-Range Expression Vectors for Single-Protein and Protein Complex Purification. Appl. Environ. Microbiol. 70: 712-721 [Abstract] [Full Text]  
• Soares, C. R.J., Gomide, F. I.C., Ueda, E. K.M., Bartolini, P. (2003). Periplasmic expression of human growth hormone via plasmid vectors containing the {lambda}PL promoter: use of HPLC for product quantification. Protein Eng Des Sel 16: 1131-1138 [Abstract] [Full Text]  
• Lueking, A., Possling, A., Huber, O., Beveridge, A., Horn, M., Eickhoff, H., Schuchardt, J., Lehrach, H., Cahill, D. J. (2003). A Nonredundant Human Protein Chip for Antibody Screening and Serum Profiling. Mol. Cell. Proteomics 2: 1342-1349 [Abstract] [Full Text]  
• Kimura, S., Iyanagi, T. (2003). High-Level Expression of Porcine Liver Cytochrome P-450 Reductase Catalytic Domain in Escherichia coli by Modulating the Predicted Local Secondary Structure of mRNA. J Biochem 134: 403-413 [Abstract] [Full Text]  
• Choi, J. H., Lee, S. J., Lee, S. J., Lee, S. Y. (2003). Enhanced Production of Insulin-Like Growth Factor I Fusion Protein in Escherichia coli by Coexpression of the Down-Regulated Genes Identified by Transcriptome Profiling. Appl. Environ. Microbiol. 69: 4737-4742 [Abstract] [Full Text]  
• Mavromatis, K., Feller, G., Kokkinidis, M., Bouriotis, V. (2003). Cold adaptation of a psychrophilic chitinase: a mutagenesis study. Protein Eng Des Sel 16: 497-503 [Abstract] [Full Text]  
• Pan, K.-L., Hsiao, H.-C., Weng, C.-L., Wu, M.-S., Chou, C. P. (2003). Roles of DegP in Prevention of Protein Misfolding in the Periplasm upon Overexpression of Penicillin Acylase in Escherichia coli. J. Bacteriol. 185: 3020-3030 [Abstract] [Full Text]  
• Woodward, L. A., Arimitsu, H., Hirst, R., Oguma, K. (2003). Expression of HC Subunits from Clostridium botulinum Types C and D and Their Evaluation as Candidate Vaccine Antigens in Mice. Infect. Immun. 71: 2941-2944 [Abstract] [Full Text]  
• Tregoning, J. S., Nixon, P., Kuroda, H., Svab, Z., Clare, S., Bowe, F., Fairweather, N., Ytterberg, J., Wijk, K. J. v., Dougan, G., Maliga, P. (2003). Expression of tetanus toxin Fragment C in tobacco chloroplasts. Nucleic Acids Res 31: 1174-1179 [Abstract] [Full Text]  
• Srinivasan, S., Barnard, G. C., Gerngross, T. U. (2002). A Novel High-Cell-Density Protein Expression System Based on Ralstonia eutropha. Appl. Environ. Microbiol. 68: 5925-5932 [Abstract] [Full Text]  
• Jeong, K. J., Lee, S. Y. (2002). Excretion of Human {beta}-Endorphin into Culture Medium by Using Outer Membrane Protein F as a Fusion Partner in Recombinant Escherichia coli. Appl. Environ. Microbiol. 68: 4979-4985 [Abstract] [Full Text]  
• Ellerman, D. A., Da Ros, V. G., Cohen, D. J., Busso, D., Morgenfeld, M. M., Cuasnicu, P. S. (2002). Expression and Structure-Function Analysis of DE, a Sperm Cysteine-Rich Secretory Protein That Mediates Gamete Fusion. Biol. Reprod. 67: 1225-1231 [Abstract] [Full Text]  
• Nicolas, P., Bize, L., Muri, F., Hoebeke, M., Rodolphe, F., Ehrlich, S. D., Prum, B., Bessieres, P. (2002). Mining Bacillus subtilis chromosome heterogeneities using hidden Markov models. Nucleic Acids Res 30: 1418-1426 [Abstract] [Full Text]  
• Masri, H. P., Cornelissen, C. N. (2002). Specific Ligand Binding Attributable to Individual Epitopes of Gonococcal Transferrin Binding Protein A. Infect. Immun. 70: 732-740 [Abstract] [Full Text]  
• Utaker, J. B., Andersen, K., Aakra, A., Moen, B., Nes, I. F. (2002). Phylogeny and Functional Expression of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from the Autotrophic Ammonia-Oxidizing Bacterium Nitrosospira sp.Isolate 40KI. J. Bacteriol. 184: 468-478 [Abstract] [Full Text]  
• Sroga, G. E., Dordick, J. S. (2001). Generation of a broad esterolytic subtilisin using combined molecular evolution and periplasmic expression. Protein Eng Des Sel 14: 929-937 [Abstract] [Full Text]  
• Rouhier, N., Gelhaye, E., Sautiere, P.-E., Brun, A., Laurent, P., Tagu, D., Gerard, J., de Fay, E., Meyer, Y., Jacquot, J.-P. (2001). Isolation and Characterization of a New Peroxiredoxin from Poplar Sieve Tubes That Uses Either Glutaredoxin or Thioredoxin as a Proton Donor. Plant Physiol. 127: 1299-1309 [Abstract] [Full Text]  
• Hashimoto, Y., Yamamoto, T., Fujiwara, S., Takagi, M., Imanaka, T. (2001). Extracellular Synthesis, Specific Recognition, and Intracellular Degradation of Cyclomaltodextrins by the Hyperthermophilic Archaeon Thermococcus sp. Strain B1001. J. Bacteriol. 183: 5050-5057 [Abstract] [Full Text]  
• Short, M.K., Jeffrey, P.D., Demirjian, A., Margolies, M.N. (2001). A single H:CDR3 residue in the anti-digoxin antibody 26-10 modulates specificity for C16-substituted digoxin analogs. Protein Eng Des Sel 14: 287-296 [Abstract] [Full Text]  
• Lutz, K. A., Knapp, J. E., Maliga, P. (2001). Expression of bar in the Plastid Genome Confers Herbicide Resistance. Plant Physiol. 125: 1585-1590 [Abstract] [Full Text]  
• Kuroda, H., Maliga, P. (2001). Complementarity of the 16S rRNA penultimate stem with sequences downstream of the AUG destabilizes the plastid mRNAs. Nucleic Acids Res 29: 970-975 [Abstract] [Full Text]  
• Kuroda, H., Maliga, P. (2001). Sequences Downstream of the Translation Initiation Codon Are Important Determinants of Translation Efficiency in Chloroplasts. Plant Physiol. 125: 430-436 [Abstract] [Full Text]  
• Lu, C., Fedoroff, N. (2000). A Mutation in the Arabidopsis HYL1 Gene Encoding a dsRNA Binding Protein Affects Responses to Abscisic Acid, Auxin, and Cytokinin. Plant Cell 12: 2351-2366 [Abstract] [Full Text]  
• Zhao, X., Singh, M., Malashkevich, V. N., Kim, P. S. (2000). Structural characterization of the human respiratory syncytial virus fusion protein core. Proc. Natl. Acad. Sci. USA 10.1073/pnas.260499197v1 [Abstract] [Full Text]  
• Kurokawa, Y., Yanagi, H., Yura, T. (2000). Overexpression of Protein Disulfide Isomerase DsbC Stabilizes Multiple-Disulfide-Bonded Recombinant Protein Produced and Transported to the Periplasm in Escherichia coli. Appl. Environ. Microbiol. 66: 3960-3965 [Abstract] [Full Text]  
• Morawski, B., Segura, A., Ornston, L. N. (2000). Substrate Range and Genetic Analysis of Acinetobacter Vanillate Demethylase. J. Bacteriol. 182: 1383-1389 [Abstract] [Full Text]  
• Saribas, A. S., Mandaci, S., Daldal, F. (1999). An Engineered Cytochrome b6c1 Complex with a Split Cytochrome b Is Able To Support Photosynthetic Growth of Rhodobacter capsulatus. J. Bacteriol. 181: 5365-5372 [Abstract] [Full Text]  
• Orr, N., Galen, J. E., Levine, M. M. (1999). Expression and Immunogenicity of a Mutant Diphtheria Toxin Molecule, CRM197, and Its Fragments in Salmonella typhi Vaccine Strain CVD 908-htrA. Infect. Immun. 67: 4290-4294 [Abstract] [Full Text]  
• Izumi, M., Fujiwara, S., Takagi, M., Kanaya, S., Imanaka, T. (1999). Isolation and Characterization of a Second Subunit of Molecular Chaperonin from Pyrococcus kodakaraensis KOD1: Analysis of an ATPase-Deficient Mutant Enzyme. Appl. Environ. Microbiol. 65: 1801-1805 [Abstract] [Full Text]  
• Schwartz, T., Lowenhaupt, K., Kim, Y.-G., Li, L., Brown, B. A. II, Herbert, A., Rich, A. (1999). Proteolytic Dissection of Zab, the Z-DNA-binding Domain of Human ADAR1. J. Biol. Chem. 274: 2899-2906 [Abstract] [Full Text]  
• Ortiz, B. L., Díez, S., Urán, M. E., Rivas, J. M., Romero, M., Caicedo, V., Restrepo, A., McEwen, J. G. (1998). Use of the 27-Kilodalton Recombinant Protein from Paracoccidioides brasiliensis in Serodiagnosis of Paracoccidioidomycosis. CVI 5: 826-830 [Abstract] [Full Text]  
• Karunakaran, P., Blatny, J. M., Ertesvåg, H., Valla, S. (1998). Species-Dependent Phenotypes of Replication-Temperature-Sensitive trfA Mutants of Plasmid RK2: a Codon-Neutral Base Substitution Stimulates Temperature Sensitivity by Leading to Reduced Levels of trfA Expression. J. Bacteriol. 180: 3793-3798 [Abstract] [Full Text]  
• Ghrist, A. C., Stauffer, G. V. (1998). Promoter Characterization and Constitutive Expression of the Escherichia coli gcvR Gene. J. Bacteriol. 180: 1803-1807 [Abstract] [Full Text]  
• Kirner, S., Hammer, P. E., Hill, D. S., Altmann, A., Fischer, I., Weislo, L. J., Lanahan, M., van Pée, K.-H., Ligon, J. M. (1998). Functions Encoded by Pyrrolnitrin Biosynthetic Genes from Pseudomonas fluorescens. J. Bacteriol. 180: 1939-1943 [Abstract] [Full Text]  
• Jensen, P. R., Hammer, K. (1998). The Sequence of Spacers between the Consensus Sequences Modulates the Strength of Prokaryotic Promoters. Appl. Environ. Microbiol. 64: 82-87 [Abstract] [Full Text]  
• Lange, T. (1997). Cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in Escherichia coli. Proc. Natl. Acad. Sci. USA 94: 6553-6558 [Abstract] [Full Text]  
• Berjanskii, M. V., Riley, M. I., Xie, A., Semenchenko, V., Folk, W. R., Van Doren, S. R. (2000). NMR Structure of the N-terminal J Domain of Murine Polyomavirus T Antigens. IMPLICATIONS FOR DnaJ-LIKE DOMAINS AND FOR MUTATIONS OF T ANTIGENS. J. Biol. Chem. 275: 36094-36103 [Abstract] [Full Text]  
• Kurokawa, Y., Yanagi, H., Yura, T. (2001). Overproduction of Bacterial Protein Disulfide Isomerase (DsbC) and Its Modulator (DsbD) Markedly Enhances Periplasmic Production of Human Nerve Growth Factor in Escherichia coli. J. Biol. Chem. 276: 14393-14399 [Abstract] [Full Text]  
• Zhao, X., Singh, M., Malashkevich, V. N., Kim, P. S. (2000). Structural characterization of the human respiratory syncytial virus fusion protein core. Proc. Natl. Acad. Sci. USA 97: 14172-14177 [Abstract] [Full Text]