Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications

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Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications

Abstract

A simple and cost effective method to independently and stereo-specifically incorporate [1H,13C]-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 [1H,13C]-methyl labeling separately at Leu or Val amino acids. This is the first method that achieves Leu-specific stereospecific [1H,13C]-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-[4-13C,2,3-2H2,15N]-Thr reagent. The Thr-specific strain allows for the production of Thr-[13CH3]γ2 labeled protein with an optimal isotope incorporation using up to 50Â*% less labeled Thr than the traditional E. coli strain without the need for 2H-glycine to prevent scrambling.



Source: Journal of Biomolecular NMR