Skip to main content
SpringerLink
Log in

Protein Expression Optimization Strategies in E. coli: A Tailored Approach in Strain Selection and Parallelizing Expression Conditions

  • Protocol
  • First Online:
Insoluble Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2406))

Abstract

Escherichia coli remains a traditional and widely used host organism for recombinant protein production. Its well-studied genome, availability of vectors and strains, cheap and relatively straight-forward cultivation methods paired with reported high protein yields are reasons why E. coli is often the first-choice host expression system for recombinant protein production. The chapter enclosed here details protocols and design strategies in strain selection and methods on how to parallelize expression conditions to optimize for soluble target protein expression in E. coli. The methods described have been validated in a protein production research facility.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 149.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Fernandez FJ, Vega MC (2016) Choose a suitable expression host: a survey of available protein production platforms. Adv Exp Med Biol 896:15–24

    Article  CAS  PubMed  Google Scholar 

  2. Sezonov G, Joseleau-Petit D, D'Ari R (2007) Escherichia coli physiology in Luria-Bertani broth. J Bacteriol 189(23):8746–8749

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Soini J, Ukkonen K, Neubauer P (2008) High cell density media for Escherichia coli are generally designed for aerobic cultivations—consequences for large-scale bioprocesses and shake flask cultures. Microb Cell Factories 7:26

    Article  CAS  Google Scholar 

  4. Choi JH, Keum KC, Lee SY (2006) Production of recombinant proteins by high cell density culture of Escherichia coli. Chem Eng Sci 61(3):876–885

    Article  CAS  Google Scholar 

  5. Yang Z, Zhang L, Zhang Y, Zhang T, Feng Y, Lu X, Lan W, Wang J, Wu H, Cao C, Wang X (2011) Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method. PLoS One 6(7):e22981

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Singh A, Upadhyay V, Upadhyay AK, Singh SM, Panda AK (2015) Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process. Microb Cell Factories 14:41

    Article  CAS  Google Scholar 

  7. Singh SM, Panda AK (2005) Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng 99(4):303–310

    Article  CAS  PubMed  Google Scholar 

  8. Honda J, Andou H, Mannen T, Sugimoto S (2000) Direct refolding of recombinant human growth differentiation factor 5 for large-scale production process. J Biosci Bioeng 89(6):582–589

    Article  CAS  PubMed  Google Scholar 

  9. Sakono M, Goto M, Furusaki S (2000) Refolding of cytochrome c using reversed micelles. J Biosci Bioeng 89(5):458–462

    Article  CAS  PubMed  Google Scholar 

  10. Paraskevopoulou V, Falcone FH (2018) Polyionic tags as enhancers of protein solubility in recombinant protein expression. Microorganisms 6(2):47

    Article  CAS  PubMed Central  Google Scholar 

  11. Costa S, Almeida A, Castro A, Domingues L (2014) Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system. Front Microbiol 5:63

    PubMed  PubMed Central  Google Scholar 

  12. Zhou P, Wagner G (2010) Overcoming the solubility limit with solubility-enhancement tags: successful applications in biomolecular NMR studies. J Biomol NMR 46(1):23–31

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Quax TE, Claassens NJ, Soll D, van der Oost J (2015) Codon bias as a means to fine-tune gene expression. Mol Cell 59(2):149–161

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Gustafsson C, Govindarajan S, Minshull J (2004) Codon bias and heterologous protein expression. Trends Biotechnol 22(7):346–353

    Article  CAS  PubMed  Google Scholar 

  15. Studier FW, Moffatt BA (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189(1):113–130

    Article  CAS  PubMed  Google Scholar 

  16. Miroux B, Walker JE (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(3):289–298

    Article  CAS  PubMed  Google Scholar 

  17. Zhang X, Studier FW (1997) Mechanism of inhibition of bacteriophage T7 RNA polymerase by T7 lysozyme. J Mol Biol 269(1):10–27

    Article  CAS  PubMed  Google Scholar 

  18. Grunberg-Manago M (1999) Messenger RNA stability and its role in control of gene expression in bacteria and phages. Annu Rev Genet 33:193–227

    Article  CAS  PubMed  Google Scholar 

  19. Kido M, Yamanaka K, Mitani T, Niki H, Ogura T, Hiraga S (1996) RNase E polypeptides lacking a carboxyl-terminal half suppress a mukB mutation in Escherichia coli. J Bacteriol 178(13):3917–3925

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Lopez PJ, Marchand I, Joyce SA, Dreyfus M (1999) The C-terminal half of RNase E, which organizes the Escherichia coli degradosome, participates in mRNA degradation but not rRNA processing in vivo. Mol Microbiol 33(1):188–199

    Article  CAS  PubMed  Google Scholar 

  21. Baca AM, Hol WG (2000) Overcoming codon bias: a method for high-level overexpression of plasmodium and other AT-rich parasite genes in Escherichia coli. Int J Parasitol 30(2):113–118

    Article  CAS  PubMed  Google Scholar 

  22. Sorensen HP, Sperling-Petersen HU, Mortensen KK (2003) Production of recombinant thermostable proteins expressed in Escherichia coli: completion of protein synthesis is the bottleneck. J Chromatogr B Analyt Technol Biomed Life Sci 786(1–2):207–214

    Article  CAS  PubMed  Google Scholar 

  23. Ferrer M, Chernikova TN, Yakimov MM, Golyshin PN, Timmis KN (2003) Chaperonins govern growth of Escherichia coli at low temperatures. Nat Biotechnol 21(11):1266–1267

    Article  CAS  PubMed  Google Scholar 

  24. Bessette PH, Aslund F, Beckwith J, Georgiou G (1999) Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm. Proc Natl Acad Sci U S A 96(24):13703–13708

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Prinz WA, Aslund F, Holmgren A, Beckwith J (1997) The role of the thioredoxin and glutaredoxin pathways in reducing protein disulfide bonds in the Escherichia coli cytoplasm. J Biol Chem 272(25):15661–15667

    Article  CAS  PubMed  Google Scholar 

  26. Lobstein J, Emrich CA, Jeans C, Faulkner M, Riggs P, Berkmen M (2012) SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. Microb Cell Factories 11:56

    Article  CAS  Google Scholar 

  27. Wagner S, Klepsch MM, Schlegel S, Appel A, Draheim R, Tarry M, Hogbom M, van Wijk KJ, Slotboom DJ, Persson JO, de Gier JW (2008) Tuning Escherichia coli for membrane protein overexpression. Proc Natl Acad Sci U S A 105(38):14371–14376

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Schlegel S, Lofblom J, Lee C, Hjelm A, Klepsch M, Strous M, Drew D, Slotboom DJ, de Gier JW (2012) Optimizing membrane protein overexpression in the Escherichia coli strain Lemo21(DE3). J Mol Biol 423(4):648–659

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Protein Expression Facility, The University of Queensland, Brisbane, QLD, Australia

    Shyn Ric Tang, Balaji Somasundaram & Linda H. L. Lua

Authors
  1. Shyn Ric Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Balaji Somasundaram
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Linda H. L. Lua
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Linda H. L. Lua .

Editor information

Editors and Affiliations

  1. Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Caldes de Montbui, Spain

    Elena Garcia Fruitós

  2. Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Caldes de Montbui, Spain

    Anna Arís Giralt

Rights and permissions

Reprints and permissions

Copyright information

© 2022 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Tang, S.R., Somasundaram, B., Lua, L.H.L. (2022). Protein Expression Optimization Strategies in E. coli: A Tailored Approach in Strain Selection and Parallelizing Expression Conditions. In: Garcia Fruitós, E., Arís Giralt, A. (eds) Insoluble Proteins. Methods in Molecular Biology, vol 2406. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1859-2_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1859-2_5

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1858-5

  • Online ISBN: 978-1-0716-1859-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation