Abstract
Escherichia coli has been the workhorse for the production of recombinant proteins (1,2). However, problems often occur in recovering substantial yields of correctly folded proteins. E. coli cannot produce some proteins containing complex disulfide bonds or mammalian proteins that require posttranslational modification for activity. Overexpressed proteins are often produced in the form of inclusion bodies, from which biologically active proteins can only be recovered by complicated and costly denaturation and refolding processes. Various techniques have been developed to solve these problems, including the use of different promoters to regulate the level of expression, using different host strains, coexpression of chaperones, reduction of culture temperature, and secretion of proteins into the periplasmic space (see Fig. 1).
Strategies for the production of recombinant proteins in E. coli. Arrow A can be achieved by one of the methods described for the conversion of “Not secreted” to “Efficient secretion.”
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Lee, S. Y. (1996) High cell density cultivation of Escherichia coli. Trends Biotechnol. 14, 98–105.
Makrides, S. C. (1996) Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol. Rev. 60, 512–538.
Choi, J. H. and Lee, S. Y. (2004) Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl. Microbiol. Biotechnol. 64, 625–635.
Park, S. J., Georgiou, G., and Lee, S. Y. (1999) Secretory production of recombinant protein by a high cell density culture of a protease negative mutant Escherichia coli. Biotechnol. Prog. 15, 164–167.
Pugsley, A. P. (1993) The complete general secretory pathway in gram-negative bacteria. Microbiol. Rev. 57, 50–108.
Jeong, K. J. and Lee, S. Y. (1999) High-level production of human leptin by fed-batch cultivation of recombinant Escherichia coli and its purification. Appl. Environ. Microbiol. 65, 3027–3032.
Jeong, K. J. and Lee, S. Y. (2000) Secretory production of human leptin in Escherichia coli. Biotechnol. Bioeng. 67, 398–407.
Jeong, K. J. and Lee, S. Y. (2001) Secretory production of human granulocyte colonystimulating factor in Escherichia coli. Protein Expr. Purif. 23, 311–318.
Choi, J. H., Jeong, K. J., Kim, S. C., and Lee, S. Y. (2000) Efficient secretory production of alkaline phosphatase by high cell density culture of recombinant Escherichia coli using the Bacillus sp. Endoxylanase signal sequence. Appl. Microbiol. Biotechnol. 53, 640–645.
Jeong, K. J., Lee, P. C., Park, I. Y., Kim, M. S., and Kim, S. C. (1998) Molecular cloning and characterization of an endoxylanase gene of Bacillus sp. in Escherichia coli. Enzyme Microb. Technol. 22, 599–605.
Yim, S. C., Jeong, K. J., Chang, H. N., and Lee, S. Y. (2001) High level secretory production of human G-CSF by fed-batch culture of recombinant Escherichia coli. Bioproc. Biosyst. Eng. 24, 249–254.
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.
Jeong, K. J. and Lee, S. Y. (2000) Secretory production of human leptin in Bacillus subtilis. J. Microbiol. Biotechnol. 10, 753–758.
Kurokawa, Y., Yanagi, H., and 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.
Wulfing, C. and Rappuoli, R. (1997) Efficient production of heat-labile enterotoxin mutant proteins by overexpression of dsbA in a degP-deficient Escherichia coli strain. Arch. Microbiol. 167, 280–283.
Qiu, J., Swartz, J. R., and Georgiou, G. (1998) Expression of active human tissue-type plasminogen activator in Escherichia coli. Appl. Environ. Microbiol. 64, 4891–4896.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2005 Humana Press Inc.
About this protocol
Cite this protocol
Lee, S.Y., Choi, J.H., Lee, S.J. (2005). Secretory Production of Therapeutic Proteins in Escherichia coli . In: Smales, C.M., James, D.C. (eds) Therapeutic Proteins. Methods in Molecular Biology™, vol 308. Humana Press. https://doi.org/10.1385/1-59259-922-2:031
Download citation
DOI: https://doi.org/10.1385/1-59259-922-2:031
Publisher Name: Humana Press
Print ISBN: 978-1-58829-390-9
Online ISBN: 978-1-59259-922-6
eBook Packages: Springer Protocols