[NMR paper] Improved Protocol for the Production of the Low-Expression Eukaryotic Membrane Protein Human Aquaporin 2 in Pichia pastoris for Solid-State NMR.
Related ArticlesImproved Protocol for the Production of the Low-Expression Eukaryotic Membrane Protein Human Aquaporin 2 in Pichia pastoris for Solid-State NMR.
Biomolecules. 2020 Mar 11;10(3):
Authors: Munro R, de Vlugt J, Ladizhansky V, Brown LS
Abstract
Solid-state nuclear magnetic resonance (SSNMR) is a powerful biophysical technique for studies of membrane proteins; it requires the incorporation of isotopic labels into the sample. This is usually accomplished through over-expression of the protein of interest in a prokaryotic or eukaryotic host in minimal media, wherein all (or some) carbon and nitrogen sources are isotopically labeled. In order to obtain multi-dimensional NMR spectra with adequate signal-to-noise ratios suitable for in-depth analysis, one requires high yields of homogeneously structured protein. Some membrane proteins, such as human aquaporin 2 (hAQP2), exhibit poor expression, which can make producing a sample for SSNMR in an economic fashion extremely difficult, as growth in minimal media adds additional strain on expression hosts. We have developed an optimized growth protocol for eukaryotic membrane proteins in the methylotrophic yeast Pichia pastoris. Our new growth protocol uses the combination of sorbitol supplementation, higher cell density, and low temperature induction (LT-SEVIN), which increases the yield of full-length, isotopically labeled hAQP2 ten-fold. Combining mass spectrometry and SSNMR, we were able to determine the nature and the extent of post-translational modifications of the protein. The resultant protein can be functionally reconstituted into lipids and yields excellent resolution and spectral coverage when analyzed by two-dimensional SSNMR spectroscopy.
PMID: 32168846 [PubMed - as supplied by publisher]
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 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)...
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06-05-2018 01:31 PM
[NMR paper] Sparse (13)C labelling for solid-state NMR studies of P. pastoris expressed eukaryotic seven-transmembrane proteins.
Sparse (13)C labelling for solid-state NMR studies of P. pastoris expressed eukaryotic seven-transmembrane proteins.
Related Articles Sparse (13)C labelling for solid-state NMR studies of P. pastoris expressed eukaryotic seven-transmembrane proteins.
J Biomol NMR. 2016 Apr 27;
Authors: Liu J, Liu C, Fan Y, Munro RA, Ladizhansky V, Brown LS, Wang S
Abstract
We demonstrate a novel sparse (13)C labelling approach for methylotrophic yeast P. pastoris expression system, towards solid-state NMR studies of eukaryotic membrane proteins....
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04-29-2016 06:31 PM
Sparse 13 C labelling for solid-state NMR studies of P. pastoris expressed eukaryotic seven-transmembrane proteins
Sparse 13 C labelling for solid-state NMR studies of P. pastoris expressed eukaryotic seven-transmembrane proteins
Abstract
We demonstrate a novel sparse 13C labelling approach for methylotrophic yeast P. pastoris expression system, towards solid-state NMR studies of eukaryotic membrane proteins. The labelling scheme was achieved by co-utilizing natural abundance methanol and specifically 13C labelled glycerol as carbon sources in the expression medium. This strategy improves the spectral resolution by 1.5 fold, displays site-specific labelling...
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04-28-2016 02:15 PM
[NMR paper] Yeast-expressed human membrane protein aquaporin-1 yields excellent resolution of solid-state MAS NMR spectra.
Yeast-expressed human membrane protein aquaporin-1 yields excellent resolution of solid-state MAS NMR spectra.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--production.springer.de-OnlineResources-Logos-springerlink.gif Related Articles Yeast-expressed human membrane protein aquaporin-1 yields excellent resolution of solid-state MAS NMR spectra.
J Biomol NMR. 2013 Jan 24;
Authors: Emami S, Fan Y, Munro R, Ladizhansky V, Brown LS
Abstract
One of the biggest challenges in solid-state NMR studies of membrane proteins is to obtain a...
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02-03-2013 10:19 AM
[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 in Pichia pastoris and characterization by circular dichroism and NMR of r
Expression in Pichia pastoris and characterization by circular dichroism and NMR of rhodostomin.
Related Articles Expression in Pichia pastoris and characterization by circular dichroism and NMR of rhodostomin.
Proteins. 2001 Jun 1;43(4):499-508
Authors: Guo RT, Chou LJ, Chen YC, Chen CY, Pari K, Jen CJ, Lo SJ, Huang SL, Lee CY, Chang TW, Chaung WJ
Rhodostomin (Rho) is a snake venom protein isolated from Calloselasma rhodostoma. Rho is a disintegrin that inhibits platelet aggregation by blocking the binding of fibrinogen to the integrin...
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11-19-2010 08:32 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...
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11-19-2010 08:29 PM
[NMR paper] High-level expression of uniformly 15N-labeled hen lysozyme in Pichia pastoris and id
High-level expression of uniformly 15N-labeled hen lysozyme in Pichia pastoris and identification of the site in hen lysozyme where phosphate ion binds using NMR measurements.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--linkinghub.elsevier.com-ihub-images-PubMedLink.gif Related Articles High-level expression of uniformly 15N-labeled hen lysozyme in Pichia pastoris and identification of the site in hen lysozyme where phosphate ion binds using NMR measurements.
FEBS Lett. 1999 Apr 1;448(1):33-7
Authors: Mine S, Ueda T, Hashimoto Y, Tanaka Y,...
Improved Protocol for the Production of the Low-Expression Eukaryotic Membrane Protein Human Aquaporin 2 in Pichia pastoris for Solid-State NMR.
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