Cold-Shock Expression System in E. coli for Protein NMR Studies
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 wordsProtein expression system Escherichia coli (E. coli) pCold-GST vector Isotope labeling Single protein production (SPP) system Nuclear magnetic resonance (NMR)
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.