Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications.
Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications.
J Biomol NMR. 2016 Jun 2;
Authors: Monneau YR, Ishida Y, Rossi P, Saio T, Tzeng SR, Inouye M, Kalodimos CG
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 ?-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](?2) 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.
PMID: 27255761 [PubMed - as supplied by publisher]
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