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Protein NMR Techniques

Volume 831 of the series Methods in Molecular Biology pp 1-18

Date:

A Novel Bacterial Expression Method with Optimized Parameters for Very High Yield Production of Triple-Labeled Proteins

  • Victoria MurrayAffiliated withDepartment of Biochemistry and Molecular Biology, School of Medicine, Wayne State University
  • , Yuefei HuangAffiliated withDepartment of Biochemistry and Molecular Biology, School of Medicine, Wayne State University
  • , Jianglei ChenAffiliated withDepartment of Biochemistry and Molecular Biology, School of Medicine, Wayne State University
  • , Jianjun WangAffiliated withDepartment of Biochemistry and Molecular Biology, School of Medicine, Wayne State University
  • , Qianqian LiAffiliated withDepartment of Biochemistry and Molecular Biology, School of Medicine, Wayne State University Email author 

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Abstract

The Gram-negative bacterium Escherichia coli offer a means for rapid, high-yield, and economical production of recombinant proteins. However, when preparing protein samples for NMR, high-level production of functional isotopically labeled proteins can be quite challenging. This is especially true for the preparation of triple-labeled protein samples in D2O (2H/13C/15N). The large expense and time-consuming nature of triple-labeled protein production for NMR led us to revisit the current bacterial protein expression protocols. Our goal was to develop an efficient bacterial expression method for very high-level production of triple-labeled proteins that could be routinely utilized in every NMR lab without changing expression vectors or requiring fermentation. We developed a novel high cell-density IPTG-induction bacterial expression method that combines tightly controlled traditional IPTG-induction expression with the high cell-density of auto-induction expression. In addition, we optimize several key experimental protocols and parameters to ensure that our new high cell-density bacterial expression method routinely produces 14–25 mg of triple-labeled proteins and 15–35 mg of unlabeled proteins from 50-mL bacterial cell cultures.

Key words

High yield protein production Bacterial expression Isotopic labeling NMR