A novel bacterial expression method with optimized parameters for very high yield production of triple-labeled proteins.

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 D(2)O ((2)H/(13)C/(15)N). 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.