Advertisement

High-Throughput Production of Proteins in E. coli for Structural Studies

  • Charikleia Black
  • John J. Barker
  • Richard B. Hitchman
  • Hok Sau Kwong
  • Sam Festenstein
  • Thomas B. Acton
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1586)

Abstract

We have developed a standardized and efficient workflow for high-throughput (HT) protein expression in E. coli and parallel purification which can be tailored to the downstream application of the target proteins. It includes a one-step purification for the purposes of functional assays and a two-step protocol for crystallographic studies, with the option of on-column tag removal.

Key words

Protein purification Parallel purification High-throughput ÄKTA-Xpress E. coli expression Expression triage 

References

  1. 1.
    Braun P, LaBaer J (2003) High throughput protein production for functional proteomics. Trends Biotechnol 21:383–388CrossRefPubMedGoogle Scholar
  2. 2.
    Quevillon-Cheruel S, Collinet B, Trésaugues L et al (2006) Cloning, production, and purification of proteins for a medium-scale structural genomics project. Methods Mol Biol 363:21–38CrossRefGoogle Scholar
  3. 3.
    Saez NJ, Vincentelli R (2014) High-throughput expression screening and purification of recombinant proteins in Escherichia coli. Methods Mol Biol 1091:33–53CrossRefPubMedGoogle Scholar
  4. 4.
    Stevens RC (2000) Design of high-throughput of protein production for structural biology. Structure 8:R177–R185CrossRefPubMedGoogle Scholar
  5. 5.
    Gräslund S, Nordlund P, Weigelt J et al (2008) Protein production and purification. Nat Methods 5:135–146CrossRefPubMedGoogle Scholar
  6. 6.
    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
  7. 7.
    Shih YP, Kung WM, Chen JC et al (2002) High-throughput screening of soluble recombinant proteins. Protein Sci 11:1714–1719CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Murthy TVS, Shi Z, Fernandez M et al (2005) Automated high-throughput protein purification with MagneHis™ particles. Promega Notes 91:2–5Google Scholar
  9. 9.
    Yoo D, Provchy J, Park C et al (2014) Automated high-throughput protein purification using an ÄKTApurifier and a CETAC autosampler. J Chromatogr A 1344:23–30CrossRefPubMedGoogle Scholar
  10. 10.
    Tworak A, Podkowinski JMF (2011) High throughput protein production. J Biotechnol Comput Biol Bionanotechnol Perspect 92:119–131Google Scholar
  11. 11.
    Edwards A (2005) Purifying proteins for structural biology. In: Sundström M (ed) Structural genomics and high-throughput structural biology. Taylor and Francis, New YorkGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Charikleia Black
    • 1
  • John J. Barker
    • 1
  • Richard B. Hitchman
    • 1
  • Hok Sau Kwong
    • 1
  • Sam Festenstein
    • 1
  • Thomas B. Acton
    • 2
  1. 1.Evotec LtdOxfordshireUK
  2. 2.Evotec (U.S.) Inc.PrincetonUSA

Personalised recommendations