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

We present a simple, convenient and robust protocol for expressing perdeuterated proteins in E. coli BL21(DE3) cells in shaker flasks that reduces D 2 O usage tenfold and d 7 -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 OD 600 of up to 10. Inducing the cells with isopropyl β- d -1-thiogalactopyranoside at an OD 600 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 OD 600 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 H 2 O-based to D 2 O-based medium is also key for ensuring high levels of protein expression in D 2 O. We find that a simple three-step adaptation approach—Luria–Bertani (LB) medium in H 2 O to LB in D 2 O to modified-M9 medium in D 2 O 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.