Perdeuteration and methyl-selective 1H, 13C-labeling by using a Kluyveromyces lactis expression system

Abstract

The production of stable isotope-labeled proteins is critical in structural analyses of large molecular weight proteins using NMR. Although prokaryotic expression systems using Escherichia coli have been widely used for this purpose, yeast strains have also been useful for the expression of functional eukaryotic proteins. Recently, we reported a cost-effective stable isotope-labeled protein expression using the hemiascomycete yeast Kluyveromyces lactis (K. lactis), which allow us to express exogenous proteins at costs comparable to prokaryotic expression systems. Here, we report the successful production of highly deuterated (>90 %) protein in the K. lactis system. We also examined the methyl-selective (1)H, (13)C-labeling of Ile, Leu, and Val residues using commonly used amino acid precursors. The efficiency of (1)H-(13)C-incorporation varied significantly based on the amino acid. Although a high level of (1)H-(13)C-incorporation was observed for the Ile δ1 position, (1)H, (13)C-labeling rates of Val and Leu methyl groups were limited due to the mitochondrial localization of enzymes involved in amino acid biosynthesis and the lack of transporters for α-ketoisovalerate in the mitochondrial membrane. In line with this notion, the co-expression with branched-chain-amino-acid aminotransferase in the cytosol significantly improved the incorporation rates of amino acid precursors. Although it would be less cost-effective, addition of (13)C-labeled valine can circumvent problems associated with precursors and achieve high level (1)H, (13)C-labeling of Val and Leu. Taken together, the K. lactis system would be a good alternative for expressing large eukaryotic proteins that need deuteration and/or the methyl-selective (1)H, (13)C-labeling for the sensitive detection of NMR resonances.