Cold-Shock Expression System in E. coli for Protein NMR Studies

  • Toshihiko Sugiki
  • Toshimichi Fujiwara
  • Chojiro Kojima
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1586)

Abstract

The cold-shock system using the pCold vector is one of the most effective Escherichia coli heterologous protein expression systems. It allows the improvement of the expression level of the protein of interest in a soluble fraction. In this chapter, we describe practical procedures for the overexpression of heterologous protein of interest by using the pCold vector or the single-protein production system. The latter is one of the most advanced pCold technologies for isotope labeling of the target protein and its NMR studies.

Key words

Protein expression system Escherichia coli (E. colipCold-GST vector Isotope labeling Single protein production (SPP) system Nuclear magnetic resonance (NMR) 

Notes

Acknowledgments

We are grateful to M. Yoneyama, K. Hayashi, S. Kataoka, I. Kawahara, K. Furuita, Y. Hattori, K. Harada, S. Shinya (Osaka University, Japan), L. Mao, Y. Ishida, M. Inouye (Rutgers University, NJ), and Y. Yamaguchi (Osaka City University, Japan) for many useful suggestions and comments. This work was supported in part by KAKENHI (26840050, 26291029, 26291013, 26650018, 24000017, 24657074, 23370053) for T.S., T.F., and C.K., the Platform Project for Supporting Drug Discovery and Life Science Research, and AMED-CREST from MEXT and AMED.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Toshihiko Sugiki
    • 1
  • Toshimichi Fujiwara
    • 1
  • Chojiro Kojima
    • 1
  1. 1.Graduate School of EngineeringYokohama National UniversityYokohamaJapan

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