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

Yoan R. Monneau, Yojiro Ishida, Paolo Rossi, Tomohide Saio, Shiou Ru Tzeng, Masayori Inouye, Charalampos G. Kalodimos

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

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 50 % less labeled Thr than the traditional E. coli strain without the need for 2H-glycine to prevent scrambling.

Original languageEnglish (US)
Pages (from-to)99-108
Number of pages10
JournalJournal of biomolecular NMR
Volume65
Issue number2
DOIs
StatePublished - Jun 1 2016

Bibliographical note

Publisher Copyright:
© 2016, Springer Science+Business Media Dordrecht.

Keywords

  • Auxotrophic strains
  • Large proteins
  • Methyl labeling
  • NMR

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