A simple and robust protocol for high-yield expression of perdeuterated proteins in Escherichia coli grown in shaker...

We present a simple, convenient and robust protocol for expressing perdeuterated proteins in E. coli BL21(DE3) cells in shaker flasks that reduces D₂O usage tenfold and d₇-glucose usage by 30 %. Using a modified M9 medium and optimized growth conditions, we were able to grow cells in linear log phase to an OD₆₀₀ of up to 10. Inducing the cells with isopropyl β-D-1-thiogalactopyranoside at an OD₆₀₀ of 10, instead of less than 1, enabled us to increase the cell mass tenfold per unit volume of cell culture. We show that protein expression levels per cell are the same when induced at an OD₆₀₀ between 1 and 10 under these growth conditions. Thus, our new protocol can increase protein yield per unit volume of cell culture tenfold. Adaptation of E. coli from H₂O-based to D₂O-based medium is also key for ensuring high levels of protein expression in D₂O. We find that a simple three-step adaptation approach-Luria-Bertani (LB) medium in H₂O to LB in D₂O to modified-M9 medium in D₂O is both simple and reliable. The method increases the yield of perdeuterated proteins by up to tenfold using commonly available air shakers without any requirement for specialized fermentation equipment.