A Novel Bacterial Expression Method with Optimized Parameters for Very High Yield Production of Triple-Labeled Proteins
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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.
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- A Novel Bacterial Expression Method with Optimized Parameters for Very High Yield Production of Triple-Labeled Proteins
- Book Title
- Protein NMR Techniques
- pp 1-18
- Print ISBN
- Online ISBN
- Series Title
- Methods in Molecular Biology
- Series Volume
- Series Subtitle
- Methods and Protocols
- Series ISSN
- Humana Press
- Copyright Holder
- Springer Science+Business Media, LLC
- Additional Links
- High yield protein production
- Bacterial expression
- Isotopic labeling
- Editor Affiliations
- ID1. University of Albany, State University of New York
- ID2. University of Albany, State University of New York
- Author Affiliations
- 1. Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI, USA
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