Recombinant Protein Expression in E. coli : A Historical Perspective

Part of the Methods in Molecular Biology book series (MIMB, volume 1586)


This introductory chapter provides a brief historical survey of the key elements incorporated into commonly used E. coli-based expression systems. The highest impact in expression technology is associated with innovations that were based on extensively studied biological systems, and where the tools were widely distributed in the academic community.

Key words

E. coli Promoter Recombinant protein Protein engineering Expression vectors 


  1. 1.
    Muller-Hill B, Crapo L, Gilbert W (1968) Mutants that make more lac repressor. Proc Natl Acad Sci U S A 59:1259–1264CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Baeshen MN, Al-Hejin AM, Bora RS et al (2015) Production of biopharmaceuticals in E. coli: current scenario and future perspectives. J Microbiol Biotechnol 25:953–962CrossRefPubMedGoogle Scholar
  3. 3.
    Baneyx F (1999) Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol 10:411–421CrossRefPubMedGoogle Scholar
  4. 4.
    Remaut E, Stanssens P, Fiers W (1983) Inducible high level synthesis of mature human fibroblast interferon in Escherichia coli. Nucleic Acids Res 11:4677–4688CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Guzman LM, Belin D, Carson MJ et al (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177:4121–4130CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Brunner M, Bujard H (1987) Promoter recognition and promoter strength in the Escherichia coli system. EMBO J 6:3139–3144PubMedPubMedCentralGoogle Scholar
  7. 7.
    Deuschle U, Kammerer W, Gentz R et al (1986) Promoters of Escherichia coli: a hierarchy of in vivo strength indicates alternate structures. EMBO J 5:2987–2994PubMedPubMedCentralGoogle Scholar
  8. 8.
    Rosenberg AH, Lade BN, Chui DS et al (1987) Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene 56:125–135CrossRefPubMedGoogle Scholar
  9. 9.
    Dubendorff JW, Studier FW (1991) Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J Mol Biol 219:45–59CrossRefPubMedGoogle Scholar
  10. 10.
    Studier FW (2014) Stable expression clones and auto-induction for protein production in E. coli. Methods Mol Biol 1091:17–32CrossRefPubMedGoogle Scholar
  11. 11.
    Burgess-Brown NA, Sharma S, Sobott F et al (2008) Codon optimization can improve expression of human genes in Escherichia coli: a multi-gene study. Protein Expr Purif 59:94–102CrossRefPubMedGoogle Scholar
  12. 12.
    Munro S, Pelham HR (1984) Use of peptide tagging to detect proteins expressed from cloned genes: deletion mapping functional domains of Drosophila hsp 70. EMBO J 3:3087–3093PubMedPubMedCentralGoogle Scholar
  13. 13.
    Hopp TP, Prickett KS, Price VL et al (1988) A short polypeptide marker sequence useful for recombinant protein identification and purification. Nat Biotechnol 6:1204–1210CrossRefGoogle Scholar
  14. 14.
    Field J, Nikawa J, Broek D et al (1988) Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol Cell Biol 8:2159–2165CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Robertson D, Paterson HF, Adamson P et al (1995) Ultrastructural localization of ras-related proteins using epitope-tagged plasmids. J Histochem Cytochem 43:471–480CrossRefPubMedGoogle Scholar
  16. 16.
    Hochuli E, Dobeli H, Schacher A (1987) New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J Chromatogr 411:177–184CrossRefPubMedGoogle Scholar
  17. 17.
    Smith DB, Johnson KS (1988) Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67:31–40CrossRefPubMedGoogle Scholar
  18. 18.
    Lee SC, Olins PO (1992) Effect of overproduction of heat shock chaperones GroESL and DnaK on human procollagenase production in Escherichia coli. J Biol Chem 267:2849–2852PubMedGoogle Scholar
  19. 19.
    Nishihara K, Kanemori M, Kitagawa M et al (1998) Chaperone coexpression plasmids: differential and synergistic roles of DnaK-DnaJ-GrpE and GroEL-GroES in assisting folding of an allergen of Japanese cedar pollen, Cryj2, in Escherichia coli. Appl Environ Microbiol 64:1694–1699PubMedPubMedCentralGoogle Scholar
  20. 20.
    Ferrer M, Chernikova TN, Timmis KN et al (2004) Expression of a temperature-sensitive esterase in a novel chaperone-based Escherichia coli strain. Appl Environ Microbiol 70:4499–4504CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Bessette PH, Aslund F, Beckwith J et al (1999) Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm. Proc Natl Acad Sci U S A 96:13703–13708CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Shrestha A, Hamilton G, O'Neill E et al (2012) Analysis of conditions affecting auto-phosphorylation of human kinases during expression in bacteria. Protein Expr Purif 81:136–143CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Savitsky P, Bray J, Cooper CD et al (2010) High-throughput production of human proteins for crystallization: the SGC experience. J Struct Biol 172:3–13CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Tsai J, Lee JT, Wang W et al (2008) Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity. Proc Natl Acad Sci U S A 105:3041–3046CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Schlinkmann KM, Hillenbrand M, Rittner A et al (2012) Maximizing detergent stability and functional expression of a GPCR by exhaustive recombination and evolution. J Mol Biol 422:414–428CrossRefPubMedGoogle Scholar
  26. 26.
    Serrano-Vega MJ, Magnani F, Shibata Y et al (2008) Conformational thermostabilization of the beta1-adrenergic receptor in a detergent-resistant form. Proc Natl Acad Sci U S A 105:877–882CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Aslanidis C, de Jong PJ (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res 18:6069–6074CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Klock HE, Lesley SA (2009) The polymerase incomplete primer extension (PIPE) method applied to high-throughput cloning and site-directed mutagenesis. Methods Mol Biol 498:91–103CrossRefPubMedGoogle Scholar
  29. 29.
    Unger T, Jacobovitch Y, Dantes A et al (2010) Applications of the restriction free (RF) cloning procedure for molecular manipulations and protein expression. J Struct Biol 172:34–44CrossRefPubMedGoogle Scholar
  30. 30.
    Hartley JL, Temple GF, Brasch MA (2000) DNA cloning using in vitro site-specific recombination. Genome Res 10:1788–1795CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Bernard P, Gabant P, Bahassi EM et al (1994) Positive-selection vectors using the F plasmid ccdB killer gene. Gene 148:71–74CrossRefPubMedGoogle Scholar
  32. 32.
    Gay P, Le Coq D, Steinmetz M et al (1985) Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria. J Bacteriol 164:918–921PubMedPubMedCentralGoogle Scholar
  33. 33.
    Haffke M, Marek M, Pelosse M et al (2015) Characterization and production of protein complexes by co-expression in Escherichia coli. Methods Mol Biol 1261:63–89CrossRefPubMedGoogle Scholar
  34. 34.
    Structural Genomics C, China Structural Genomics C, Northeast Structural Genomics C et al (2008) Protein production and purification. Nat Methods 5:135–146CrossRefGoogle Scholar
  35. 35.
    Vincentelli R, Cimino A, Geerlof A et al (2011) High-throughput protein expression screening and purification in Escherichia coli. Methods 55:65–72CrossRefPubMedGoogle Scholar
  36. 36.
    Cairns J, Stent GS, Watson JD (2007) In: Centennial (ed) Phage and the origins of molecular biology. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  37. 37.
    Cong L, Ran FA, Cox D et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Mali P, Yang L, Esvelt KM et al (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Chen R (2012) Bacterial expression systems for recombinant protein production: E. coli and beyond. Biotechnol Adv 30:1102–1107CrossRefPubMedGoogle Scholar
  40. 40.
    Makino T, Skretas G, Georgiou G (2011) Strain engineering for improved expression of recombinant proteins in bacteria. Microb Cell Fact 10:32CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Hutchison CA 3rd, Chuang RY, Noskov VN et al (2016) Design and synthesis of a minimal bacterial genome. Science 351:aad6253CrossRefPubMedGoogle Scholar
  42. 42.
    Galanie S, Thodey K, Trenchard IJ et al (2015) Complete biosynthesis of opioids in yeast. Science 349:1095–1100CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Paddon CJ, Westfall PJ, Pitera DJ et al (2013) High-level semi-synthetic production of the potent antimalarial artemisinin. Nature 496:528–532CrossRefPubMedGoogle Scholar
  44. 44.
    Kamens J (2015) The Addgene repository: an international nonprofit plasmid and data resource. Nucleic Acids Res 43:D1152–D1157CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Structural Genomics ConsortiumUniversity of OxfordOxfordUK

Personalised recommendations