Auto-inducing media for uniform isotope labeling of proteins with (15)N, (13)C and (2)H

Auto-inducing media for protein expression offer many advantages like robust reproducibility, high yields of soluble protein and much reduced workload. Here, an auto-inducing medium for uniform isotope labelling of proteins with (15)N, (13)C and/or (2)H in E. coli is presented. So far, auto-inducing media have not found widespread application in the NMR field, because of the prohibitively high cost of labeled lactose, which is an essential ingredient of such media. Here, we propose using lactose that is only selectively labeled on the glucose moiety. It can be synthesized from inexpensive and readily available substrates: labeled glucose and unlabeled activated galactose. With this approach, uniformly isotope labeled proteins were expressed in unattended auto-inducing cultures with incorporation of (13)C, (15)N of 96.6% and (2)H, (15)N of 98.8%. With the present protocol, the NMR community could profit from the many advantages that auto-inducing media offer.