Recent developments of methyl-labeling strategies in Pichia pastoris for NMR spectroscopy

Abstract

Nuclear magnetic resonance (NMR) spectroscopy is a primary structural biology method to characterize protein dynamics in solution. For large macromolecular systems, methyl-labeling in a perdeuterated background significantly improves the relaxation properties, while providing sensitive probes for structure and dynamics analysis. However, how to prepare methyl-labeled proteins, especially for functional eukaryotic proteins, remains to be a major bottleneck in this field. Due to its advantages in eukaryotic co-translational and post-translational modification, as well as high-density fermentation, Pichia pastoris has been a cost-effective platform strain for ¹³C, ¹⁵N-labeling and deuterium labeling since the early 2000's. Recently, some substantial progress has been made in methyl-labeling, such as the feasibility of ¹³C isoleucine δ1 methyl-labeling in perdeuterated background and the increased uptake of the Val/Leu precursor. Here, we systematically introduce the isotope-labeling strategies in P. pastoris, including strain engineering and detailed fermentation protocols in ¹³C, ¹⁵N-labeling and methyl-labeling, providing instructions and guidance for the future improvement of sample preparation for NMR spectroscopy.

Keywords: Isotope-labeling; Komagataella phaffii; Nuclear magnetic resonance spectroscopy; Pichia pastoris.