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

Methyl-selective isotope labeling using α-ketoisovalerate for the yeast Pichia pastoris... 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 supe- rior 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 dicu ffi lty, we explored various culture conditions for the yeast cells to ec ffi iently 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 1 13 the synthesized recombinant protein yielded intense H– C correlation signals. Based on these results, we present optimized protocols for the Val/Leu-methyl-selective C incorporation by the P. pastoris expression system. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biomolecular NMR Springer Journals

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

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Publisher
Springer Netherlands
Copyright
Copyright © 2018 by Springer Science+Business Media B.V., part of Springer Nature
Subject
Physics; Biological and Medical Physics, Biophysics; Biochemistry, general; Spectroscopy/Spectrometry
ISSN
0925-2738
eISSN
1573-5001
D.O.I.
10.1007/s10858-018-0192-3
Publisher site
See Article on Publisher Site

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 supe- rior 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 dicu ffi lty, we explored various culture conditions for the yeast cells to ec ffi iently 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 1 13 the synthesized recombinant protein yielded intense H– C correlation signals. Based on these results, we present optimized protocols for the Val/Leu-methyl-selective C incorporation by the P. pastoris expression system.

Journal

Journal of Biomolecular NMRSpringer Journals

Published: Jun 5, 2018

References

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