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

• Published in: Journal of biomolecular NMR 2013-11, Vol.57 (3), p.297
• Main Authors: Miyazawa-onami, Mayumi, Takeuchi, Koh, Takano, Toshiaki, Sugiki, Toshihiko, Shimada, Ichio, Takahashi, Hideo
• Format: Article
• Language: English
• Subjects: Amino acids; Biosynthesis; E coli; Proteins; Stable isotopes; Yeasts
• ISSN: 0925-2738; 1573-5001
• DOI: 10.1007/s10858-013-9789-8

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 ^sup 1^H, ^sup 13^C-labeling of Ile, Leu, and Val residues using commonly used amino acid precursors. The efficiency of ^sup 1^H- ^sup 13^C-incorporation varied significantly based on the amino acid. Although a high level of ^sup 1^H-^sup 13^C-incorporation was observed for the Ile δ1 position, ^sup 1^H, ^sup 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 [alpha]-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 ^sup 13^C-labeled valine can circumvent problems associated with precursors and achieve high level ^sup 1^H, ^sup 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 ^sup 1^H, ^sup 13^C-labeling for the sensitive detection of NMR resonances.[PUBLICATION ABSTRACT]