Methyl-selective isotope labeling using α-ketoisovalerate for the yeast Pichia pastoris recombinant protein expression system

Abstract

Methyl-detected NMR spectroscopy is a useful tool for investigating the structures and interactions of large macromolecules such as membrane proteins. The procedures for preparation of methyl-specific isotopically-labeled proteins were established for the Escherichia coli (E. coli) expression system, but typically it is not feasible to express eukaryotic proteins using E. coli. The Pichia pastoris (P. pastoris) expression system is the most common yeast expression system, and is known to be superior to the E. coli system for the expression of mammalian proteins, including secretory and membrane proteins. However, this system has not yet been optimized for methyl-specific isotope labeling, especially for Val/Leu-methyl specific isotope incorporation. To overcome this difficulty, we explored various culture conditions for the yeast cells to efficiently uptake Val/Leu precursors. Among the searched conditions, we found that the cultivation pH has a critical effect on Val/Leu precursor uptake. At an acidic cultivation pH, the uptake of the Val/Leu precursor was increased, and methyl groups of Val and Leu in the synthesized recombinant protein yielded intense ¹H-¹³C correlation signals. Based on these results, we present optimized protocols for the Val/Leu-methyl-selective ¹³C incorporation by the P. pastoris expression system.

Keywords: Eukaryotic expression system; Methyl-selective isotope labeling; Pichia pastoris; Stable isotope labeling.