Abstract
A simple and cost effective method to independently and stereo-specifically incorporate [(1)H,(13)C]-methyls in Leu and Val in proteins is presented. Recombinant proteins for NMR studies are produced using a tailored set of auxotrophic E. coli strains. NMR active isotopes are routed to either Leu or Val methyl groups from the commercially available and scrambling-free precursors α-ketoisovalerate and acetolactate. The engineered strains produce deuterated proteins with stereospecific [(1)H,(13)C]-methyl labeling separately at Leu or Val amino acids. This is the first method that achieves Leu-specific stereospecific [(1)H,(13)C]-methyl labeling of proteins and scramble-free Val-specific labeling. Use of auxotrophs drastically decreases the amount of labeled precursor required for expression without impacting the yield. The concept is extended to Thr methyl labeling by means of a Thr-specific auxotroph that provides enhanced efficiency for use with the costly L-[4-(13)C,2,3-(2)H2,(15)N]-Thr reagent. The Thr-specific strain allows for the production of Thr-[(13)CH3](γ2) labeled protein with an optimal isotope incorporation using up to 50 % less labeled Thr than the traditional E. coli strain without the need for (2)H-glycine to prevent scrambling.