Heterologous Gene Expression in E.coli pp 195-209

Part of the Methods in Molecular Biology book series (MIMB, volume 705)

Recent Developments in Difficult Protein Expression: A Guide to E. coli Strains, Promoters, and Relevant Host Mutations

Protocol

Abstract

Escherichia coli is a versatile and popular tool for heterologous protein production. Some of the reasons for its popularity include rapid growth, a variety of portable vectors, relatively simple genetics, and the potential for high-density cultivation. In addition, the extensive laboratory use of E. coli has resulted in technologies to target protein overexpression to various intracellular compartments. This is advantageous because these compartments have different environments that may facilitate folding of particular proteins of interest. This chapter discusses the properties of many of the E. coli strains available for protein expression in order to facilitate the choice of the best expression host for a particular protein of interest.

Key words

Escherichia coli protein expression T7 promoter tac promoter membrane protein expression expression strain 

References

  1. 1.
    Gilbert, W., Müller-Hill, B. (1966) Isolation of the lac repressor. Proc Natl Acad Sci USA 56, 1891–1898.PubMedCrossRefGoogle Scholar
  2. 2.
    Calos, M. P. (1978) DNA sequence for a low-level promoter of the lac repressor gene and an ‘up’ promoter mutation. Nature 274, 762–765.PubMedCrossRefGoogle Scholar
  3. 3.
    Hirschel, B. J., Shen, V., Schlessinger, D. (1980) Lactose operon transcription from wild-type and L8-UV5 lac promoters in Escherichia coli treated with chloramphenicol. J Bacteriol 143, 1534–1537.PubMedGoogle Scholar
  4. 4.
    Eron, L., Block, R. (1971) Mechanism of initiation and repression of in vitro transcription of the lac operon of Escherichia coli. Proc Natl Acad Sci USA 68, 1828–1832.PubMedCrossRefGoogle Scholar
  5. 5.
    deBoer H. A., Comstock, L. J., Vasser, M. (1983) The tac promoter: a functional hybrid derived from trp and lac promoters. Proc Natl Acad Sci USA 80, 21–25.CrossRefGoogle Scholar
  6. 6.
    Amann, E., Brosius, J., Ptashne, M. (1983) Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene 25, 167–178.PubMedCrossRefGoogle Scholar
  7. 7.
    Mulligan, M. E., Brosius, J., Clure, W. R. (1985) Characterization in vitro of the effect of spacer length on the activity of Escherichia coli RNA polymerase at the tac promoter. J Biol Chem 260, 3529–3538.PubMedGoogle Scholar
  8. 8.
    Studier, F. W. (2005) Protein production by auto-induction in high-density shaking cultures. Protein Expr Purif 41, 207–234.PubMedCrossRefGoogle Scholar
  9. 9.
    Pan, S. H., Malcolm, B. A. (2000) Reduced background expression and improved plasmid stability with pET vectors in BL21(DE3). Biotechniques 29, 1234–1237.PubMedGoogle Scholar
  10. 10.
    Zhang, X., Studier, W. F. (1997) Mechanism of inhibition of bacteriophage T7 RNA polymerase by T7 lysozyme. J Mol Biol 269, 10–27.PubMedCrossRefGoogle Scholar
  11. 11.
    Cheng, X., Zhang, X., Pflugrath, J. W., Studier, W. F. (1994) The structure of bacteriophage T7 lysozyme, a zinc amidase and an inhibitor of T7 RNA polymerase. Proc Natl Acad Sci USA 91, 4034–4038.PubMedCrossRefGoogle Scholar
  12. 12.
    Studier, W. F., Rosenberg, A. H., Dunn, J. J., Dubendorff, J. W. (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185, 60–89.PubMedCrossRefGoogle Scholar
  13. 13.
    Haldimann, A., Daniels, L. L., Wanner, B. L. (1998) Use of new methods for construction of tightly regulated arabinose and rhamnose promoter fusions in studies of the Escherichia coli phosphate regulon. J Bacteriol 180, 1277–1286.PubMedGoogle Scholar
  14. 14.
    Miroux, B., Walker, J. E. (1996) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol 260, 289–298.PubMedCrossRefGoogle Scholar
  15. 15.
    Wagner, S., Klepsch, M. M., Schlegel, S., Appel, A., Draheim, R., Tarry, M., Högbom, M., van Wijk, K. J., Slotboom, D. J., Persson, J. O., de Gier, J. W. (2008) Tuning Escherichia coli for membrane protein overexpression. Proc Natl Acad Sci USA 105, 14371–14376.PubMedCrossRefGoogle Scholar
  16. 16.
    Suzuki, M., Mao, L., Inouye, M. (2007) Single protein production (SPP) system in Escherichia coli. Nat Protocols 2, 1802–1810.CrossRefGoogle Scholar
  17. 17.
    Giacalone, M. J., Gentile, A. M., Lovitt, B. T., Berkley, N. L., Gunderson, C. W., Surber, M. W. (2006) Toxic protein expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter system. Biotechniques 40, 355–364.PubMedCrossRefGoogle Scholar
  18. 18.
    Siegele, D. A., Hu J. C. (1997) Gene expression from plasmids containing the araBAD promoter at subsaturating inducer concentrations represents mixed populations. Proc Natl Acad Sci USA 94, 8168–8172.PubMedCrossRefGoogle Scholar
  19. 19.
    Khlebnikov, A., Datsenko, K. A., Skaug, T., Wanner, B. L., Keasling, J. D. (2001) Homogeneous expression of the P(BAD) promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter. Microbiology 147, 3241–3247.PubMedGoogle Scholar
  20. 20.
    Better, M. D. (2004) Methods and cells for expression of recombinant protein products under the transcriptional control of an inducible promoter. U.S. Patent 6,803,210 Assignee: Xoma Technology Ltd., Berkeley, CA, October 12, 2004Google Scholar
  21. 21.
    Ferrer, M., Chernikova, T. N., Yakimov, M., Golyshin, P. N., Timmis, K. N. (2003) Chaperonins govern growth of Escherichia coli at low temperatures. Nat Biotechnol 21, 1266–1267.PubMedCrossRefGoogle Scholar
  22. 22.
    Derman, A. I., Prinz, W. A. Belin, D., Beckwith, J. (1993) Mutations that allow disulfide bond formation in the cytoplasm of Escherichia coli. Science 262, 1744–1747.PubMedCrossRefGoogle Scholar
  23. 23.
    Stewart, E. J., Aslund, F., Beckwith, J. (1998) Disulfide bond formation in the Escherichia coli cytoplasm: an in vivo role reversal for the thioredoxins. EMBO J 17, 5543–5550.PubMedCrossRefGoogle Scholar
  24. 24.
    Schierle, C.F., Berkmen, M., Huber, D., Kumamoto, C., Boyd, D., Beckwith, J. (2003) The DsbA signal sequence directs efficient, cotranslational export of passenger proteins to the Escherichia coli periplasm via the signal recognition particle pathway. J Bacteriol 185, 5706–5713.PubMedCrossRefGoogle Scholar
  25. 25.
    Emanuelsson, O., Brunak, S., von Heijne, G., Nielsen, H. (2006) Locating proteins in the cell using TargetP, SignalP and related tools. Nat Protocols 2, 953–971.CrossRefGoogle Scholar
  26. 26.
    Käll, L., Krogh, A., Sonnhammer, E. L. (2007) Advantages of combined transmembrane topology and signal peptide prediction – the Phobius web server. Nucleic Acids Res 35, W429–W432.PubMedCrossRefGoogle Scholar
  27. 27.
    Grisshammer, R., Duckworth, R., Henderson, R. (1993) Expression of a rat neurotensin receptor in Escherichia coli. Biochem J 295, 571–576.PubMedGoogle Scholar
  28. 28.
    Luo, J., Choulet, J., Samuelson, J. C. (2009) Rational design of a fusion partner for membrane protein expression in E. coli. Protein Sci 18, 1735–1744.PubMedCrossRefGoogle Scholar
  29. 29.
    Neophytou, I., Harvey, R., Lawrence, J., Marsh, P., Panaretou, B., Barlow, D. (2007) Eukaryotic integral membrane protein expression utilizing the Escherichia coli glycerol-conducting channel protein (GlpF). Appl Microbiol Biotechnol 77, 375–381.PubMedCrossRefGoogle Scholar
  30. 30.
    Huber, D., Boyd, D., Xia, Y., Olma, M. H., Gerstein M., Beckwith J. (2005) Use of thioredoxin as a reporter to identify a subset of Escherichia coli signal sequences that promote signal recognition particle-dependent translocation. J Bacteriol 187, 2983–2991.PubMedCrossRefGoogle Scholar
  31. 31.
    Gardner, T. S., Cantor, C. R., Collins, J. J. (2000) Construction of a genetic toggle switch in Escherichia coli. Nature 403, 339–342.PubMedCrossRefGoogle Scholar
  32. 32.
    Tucker, J., Grisshammer, R. (1996) Purification of a rat neurotensin receptor expressed in Escherichia coli. Biochem J 317, 891–899.PubMedGoogle Scholar
  33. 33.
    Yeliseev, A. A., Wong, K. K., Soubias. O., Gawrisch, K. (2005) Expression of human peripheral cannabinoid receptor for structural studies. Protein Sci 14, 2638–2653.PubMedCrossRefGoogle Scholar
  34. 34.
    Hwang, B. Y., Varadarajan, N., Li, H., Rodriguez, S., Iverson, B. L., Georgiou, G. (2007) Substrate specificity of the Escherichia coli outer membrane protease OmpP. J Bacteriol 189, 522–530.PubMedCrossRefGoogle Scholar
  35. 35.
    Link, A. J., Skretas, G., Strauch, E.-M., Chari, N. S., Georgiou, G. (2008) Efficient production of membrane-integrated and detergent-soluble G protein-coupled receptors in Escherichia coli. Protein Sci 17, 1857–1863.PubMedCrossRefGoogle Scholar
  36. 36.
    Lewis, K. (2000) Programmed death in bacteria. Micro Mol Biol Rev 64, 503–514.CrossRefGoogle Scholar
  37. 37.
    Grossman, T. H., Kawasaki, E. S., Punreddy, S. R., Osburne, M. S. (1998) Spontaneous cAMP-dependent derepression of gene expression in stationary phase plays a role in recombinant expression instability. Gene 209, 95–103.PubMedCrossRefGoogle Scholar
  38. 38.
    Guzman, L. M., Belin, D., Carson, M. J., Beckwith, J. (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177, 4121–4130.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.New England Biolabs, Inc.IpswichUSA

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