title: 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 [(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.
authors.affiliation: Center for Integrative Proteomics Research and Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, 08854, USA.
authors.forename: Yoan R
authors.initials: YR
authors.lastname: Monneau
authors.affiliation: Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, NJ, 08854, USA.
authors.forename: Yojiro
authors.initials: Y
authors.lastname: Ishida
authors.affiliation: Center for Integrative Proteomics Research and Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, 08854, USA.|Deparment of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, 55455, USA.
authors.forename: Paolo
authors.initials: P
authors.lastname: Rossi
authors.affiliation: Center for Integrative Proteomics Research and Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, 08854, USA.
authors.forename: Tomohide
authors.initials: T
authors.lastname: Saio
authors.affiliation: Center for Integrative Proteomics Research and Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, 08854, USA.
authors.forename: Shiou-Ru
authors.initials: SR
authors.lastname: Tzeng
authors.affiliation: Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, NJ, 08854, USA. inouye@cabm.rutgers.edu.
authors.forename: Masayori
authors.initials: M
authors.lastname: Inouye
authors.affiliation: Center for Integrative Proteomics Research and Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, 08854, USA. ckalodim@umn.edu.|Deparment of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, 55455, USA. ckalodim@umn.edu.
authors.forename: Charalampos G
authors.initials: CG
authors.lastname: Kalodimos
mesh_terms: D029968:Escherichia coli Proteins / Q000737:chemistry*; D007553:Isotope Labeling; D007930:Leucine / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry*; D013912:Threonine / Q000737:chemistry*; D014633:Valine / Q000737:chemistry*
chemical_list: D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D013912:Threonine; D007930:Leucine; D014633:Valine
keywords: Auxotrophic strains; Large proteins; Methyl labeling; NMR