Resolving Bottlenecks for Recombinant Protein Expression in E. coli

Part of the Methods in Molecular Biology book series (MIMB, volume 800)

Abstract

Escherichia coli is widely used as an expression system for production of recombinant proteins of prokaryotic and eukaryotic origin. A large body of knowledge has accumulated throughout the last few decades regarding expression of recombinant proteins in E. coli. However, despite this progress, protein production, primarily of eukaryotic origin, still remains a challenge. The biggest obstacle lies in obtaining large amounts of a given protein in a correctly folded form. Several strategies are being used to increase both yield and solubility. These include expression as fusion proteins, co-expression with molecular chaperones, or with a protein partner(s), and the use of multiple constructs for each protein. In this chapter, we focus on strategies for creating expression vectors, as well as on guidelines for improving recombinant protein solubility.

Key words

Protein expression Fusion-tags Restriction-free cloning Protein solubility E. coli 

References

  1. 1.
    Baneyx F (1999) Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol 10: 411–421PubMedCrossRefGoogle Scholar
  2. 2.
    Malhotra A (2009) Tagging for protein expression. Methods Enzymol 463: 239–258PubMedCrossRefGoogle Scholar
  3. 3.
    Esposito D, Chatterjee DK (2006) Enhancement of soluble protein expression through the use of fusion tags. Curr Opin Biotechnol 17: 353–358PubMedCrossRefGoogle Scholar
  4. 4.
    Yumerefendi H, Tarendeau F, Mas PJ, Hart DJ (2010) ESPRIT: an automated, library-based method for mapping and soluble expression of protein domains from challenging targets. J Struct Biol 172: 66–74PubMedCrossRefGoogle Scholar
  5. 5.
    Thomas JG, Baneyx F (1996) Protein folding in the cytoplasm of Escherichia coli: requirements for the DnaK-DnaJ-GrpE and GroEL-GroES molecular chaperone machines. Mol Microbiol 21: 1185–1196PubMedCrossRefGoogle Scholar
  6. 6.
    Gragerov A, Nudler E, Komissarova N, Gaitanaris GA, Gottesman ME, Nikiforov V (1992) Cooperation of GroEL/GroES and DnaK/DnaJ heat shock proteins in preventing protein misfolding in Escherichia coli. Proc Natl Acad Sci USA 89: 10341–10344PubMedCrossRefGoogle Scholar
  7. 7.
    Romier C, Ben Jelloul M, Albeck S, et al (2006) Co-expression of protein complexes in prokaryotic and eukaryotic hosts: experimental procedures, database tracking and case studies. Acta Crystallogr D Biol Crystallogr 62: 1232–1242PubMedCrossRefGoogle Scholar
  8. 8.
    Blackwell JR, Horgan R (1991) A novel strategy for production of a highly expressed recombinant protein in an active form. FEBS Lett 295: 10–12PubMedCrossRefGoogle Scholar
  9. 9.
    Graslund S, Nordlund P, Weigelt J, et al (2008) Protein production and purification. Nat Methods 5: 135–146PubMedCrossRefGoogle Scholar
  10. 10.
    Gileadi O, Burgess-Brown NA, Colebrook SM, et al (2008) High throughput production of recombinant human proteins for crystallography. Methods Mol Biol 426: 221–246PubMedCrossRefGoogle Scholar
  11. 11.
    van den Ent F, Lowe J (2006) RF cloning: a restriction-free method for inserting target genes into plasmids. J Biochem Biophys Methods 67: 67–74PubMedCrossRefGoogle Scholar
  12. 12.
    Unger, T, Jacobovitch Y, Dantes A, Bernheim R, Peleg Y. (2010) Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expression. J Struct Biol 172: 34–44PubMedCrossRefGoogle Scholar
  13. 13.
    Peleg Y, Unger T (2008) Application of high-throughput methodologies to the expression of recombinant proteins in E. coli. Methods Mol Biol 426: 197–208PubMedCrossRefGoogle Scholar
  14. 14.
    Berrow NS, Bussow K, Coutard B, et al (2006) Recombinant protein expression and solubility screening in Escherichia coli: a comparative study. Acta Crystallogr D Biol Crystallogr 62: 1218–1226PubMedCrossRefGoogle Scholar
  15. 15.
    Inoue H, Nojima H, Okayama H (1990) High efficiency transformation of Escherichia coli with plasmids. Gene 96: 23–28PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Israel Structural Proteomics Center, Faculty of BiochemistryWeizmann Institute of ScienceRehovotIsrael

Personalised recommendations