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 1Hâ??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.
Deuteration and selective labeling of alanine methyl groups of β 2 -adrenergic receptor expressed in a baculovirus-insect cell expression system
Deuteration and selective labeling of alanine methyl groups of β 2 -adrenergic receptor expressed in a baculovirus-insect cell expression system
Abstract
G protein-coupled receptors (GPCRs) exist in equilibrium between multiple conformations, and their populations and exchange rates determine their functions. However, analyses of the conformational dynamics of GPCRs in lipid bilayers are still challenging, because methods for observations of NMR signals of large proteins expressed in a baculovirus-insect cell expression system (BVES) are limited....
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[NMR paper] Erratum to: Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris.
Erratum to: Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris.
Related Articles Erratum to: Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris.
J Biomol NMR. 2016 Feb 19;
Authors: Clark L, Zahm JA, Ali R, Kukula M, Bian L, Patrie SM, Gardner KH, Rosen MK, Rosenbaum DM
PMID: 26894386
[NMR paper] Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris.
Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris.
Related Articles Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris.
J Biomol NMR. 2015 May 30;
Authors: Clark L, Zahm JA, Ali R, Kukula M, Bian L, Patrie SM, Gardner KH, Rosen MK, Rosenbaum DM
Abstract
(13)C Methyl TROSY NMR spectroscopy has emerged as a powerful method for studying the dynamics of large systems such as macromolecular assemblies and membrane proteins. Specific...
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05-31-2015 01:57 PM
Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris
Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris
Abstract
13C Methyl TROSY NMR spectroscopy has emerged as a powerful method for studying the dynamics of large systems such as macromolecular assemblies and membrane proteins. Specific 13C labeling of aliphatic methyl groups and perdeuteration has been limited primarily to proteins expressed in E. coli, preventing studies of many eukaryotic proteins of physiological and biomedical significance. We demonstrate the feasibility of...
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05-29-2015 01:24 PM
Metabolic flux profiling of recombinant protein secreting Pichia pastoris ... - 7thSpace Interactive (press release)
Metabolic flux profiling of recombinant protein secreting Pichia pastoris ... - 7thSpace Interactive (press release)
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Metabolic flux profiling of recombinant protein secreting Pichia pastoris ...
7thSpace Interactive (press release)
... for improved protein production. Results: The metabolic flux distribution in the central metabolism of P. pastoris growing on a mixed feed of glucose and methanol was analyzed by Metabolic Flux Analysis (MFA) using 13C-NMR-derived constraints.
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05-08-2012 06:09 PM
[NMR paper] Recombinant expression of Ole e 6, a Cys-enriched pollen allergen, in Pichia pastoris
Recombinant expression of Ole e 6, a Cys-enriched pollen allergen, in Pichia pastoris yeast: detection of partial oxidation of methionine by NMR.
Related Articles Recombinant expression of Ole e 6, a Cys-enriched pollen allergen, in Pichia pastoris yeast: detection of partial oxidation of methionine by NMR.
Protein Expr Purif. 2004 Oct;37(2):336-43
Authors: Barral P, Tejera ML, Treviño MA, Batanero E, Villalba M, Bruix M, Rodríguez R
Olive pollen is one of the main causes of allergy in Mediterranean countries. Ole e 6, an olive pollen...
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11-24-2010 10:01 PM
[NMR paper] Expression of deuterium-isotope-labelled protein in the yeast pichia pastoris for NMR
Expression of deuterium-isotope-labelled protein in the yeast pichia pastoris for NMR studies.
Related Articles Expression of deuterium-isotope-labelled protein in the yeast pichia pastoris for NMR studies.
J Biomol NMR. 2000 Aug;17(4):337-47
Authors: Morgan WD, Kragt A, Feeney J
Deuterium isotope labelling is important for NMR studies of large proteins and complexes. Many eukaryotic proteins are difficult to express in bacteria, but can be efficiently produced in the methylotrophic yeast Pichia pastoris. In order to facilitate NMR studies of...
Methyl-selective isotope labeling using α-ketoisovalerate for the yeast Pichia pastoris recombinant protein expression system
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