Related ArticlesExpression 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 the malaria parasite merozoite surface protein-1 (MSP1) complex and its interactions with antibodies, we have investigated production of the MSP1-19 protein in P. pastoris grown in deuterated media. The resulting deuteration patterns were analyzed by NMR and mass spectrometry. We have compared growth characteristics and levels of heterologous protein expression in cells adapted to growth in deuterated media (95% D2O), compared with expression in non-adapted cells. We have also compared the relative deuteration levels and the distribution pattern of residual protiation in protein from cells grown either in 95% D2O medium with protiated methanol as carbon source, or in 95% D2O medium containing deuterated methanol. A high level of uniform Calpha deuteration was demonstrated, and the consequent reduction of backbone amide signal linewidths in [1H/15N]-correlation experiments was measured. Residual protiation at different positions in various amino acid residues. including the distribution of methyl isotopomers, was also investigated. The deuteration procedures examined here should facilitate economical expression of 2H/13C/15N-labelled protein samples for NMR studies of the structure and interactions of large proteins and protein complexes.
Overproduction of 15N-labeled r-RGD-hirudin in pichia pastoris for NMR studies.
Overproduction of 15N-labeled r-RGD-hirudin in pichia pastoris for NMR studies.
Overproduction of 15N-labeled r-RGD-hirudin in pichia pastoris for NMR studies.
Protein Pept Lett. 2010;17(10):1228-33
Authors: Wang J, Zhang Y, Li S, Liu X, Yan X, Song H, Yu M, Dai L, Mo W
The novel recombinant hirudin, r-RGD-hirudin, inhibits thrombin and platelet aggregation. Here, we reported over-expression of (15)N-labeled r-RGD-hirudin by Pichia pastoris in minimal medium. After extensive optimization, the yield of active r-RGD-hirudin reached ?600 mg/L when...
nmrlearner
Proteins
0
01-22-2011 01:52 PM
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment
Abstract Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging. On the one hand, difficulties with achieving proper folding, membrane insertion, and native-like post-translational modifications frequently disqualify bacterial expression systems. On the other hand, eukaryotic cell cultures can be prohibitively expensive. One of the viable alternatives,...
nmrlearner
Proteins
0
01-22-2011 03:46 AM
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.
J Biomol NMR. 2011 Jan 19;
Authors: Fan Y, Shi L, Ladizhansky V, Brown LS
Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging. On the one hand, difficulties...
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Journal club
0
01-21-2011 01:22 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, Trevińo 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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Journal club
0
11-24-2010 10:01 PM
[NMR paper] Expression, purification, and isotope labeling of a gp120 V3 peptide and production o
Expression, purification, and isotope labeling of a gp120 V3 peptide and production of a Fab from a HIV-1 neutralizing antibody for NMR studies.
Related Articles Expression, purification, and isotope labeling of a gp120 V3 peptide and production of a Fab from a HIV-1 neutralizing antibody for NMR studies.
Protein Expr Purif. 2002 Apr;24(3):374-83
Authors: Sharon M, Görlach M, Levy R, Hayek Y, Anglister J
Most human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies in infected individuals and in immunized animals are directed...
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Journal club
0
11-24-2010 08:49 PM
[NMR paper] Yeast expression and NMR analysis of the extracellular domain of muscle nicotinic ace
Yeast expression and NMR analysis of the extracellular domain of muscle nicotinic acetylcholine receptor alpha subunit.
Related Articles Yeast expression and NMR analysis of the extracellular domain of muscle nicotinic acetylcholine receptor alpha subunit.
J Biol Chem. 2002 Apr 12;277(15):12613-21
Authors: Yao Y, Wang J, Viroonchatapan N, Samson A, Chill J, Rothe E, Anglister J, Wang ZZ
The alpha subunit of the nicotinic acetylcholine receptor (AChR) from Torpedo electric organ and mammalian muscle contains high affinity binding sites for...
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Journal club
0
11-24-2010 08:49 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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Journal club
0
11-19-2010 08:32 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,...
Expression of deuterium-isotope-labelled protein in the yeast pichia pastoris for NMR
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