Document Detail


Affinity purification and characterization of a G-protein coupled receptor, Saccharomyces cerevisiae Ste2p.
MedLine Citation:
PMID:  17646109     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
We present an example of expression and purification of a biologically active G-protein coupled receptor (GPCR) from yeast. An expression vector was constructed to encode the Saccharomyces cerevisiae GPCR alpha-factor receptor (Ste2p, the STE2 gene product) containing a 9-amino acid sequence of rhodopsin that served as an epitope/affinity tag. In the construct, two glycosylation sites and two cysteine residues were removed to aid future structural and functional studies. The receptor was expressed in yeast cells and was detected as a single band in a western blot indicating the absence of glycosylation. Ligand binding and signaling assays of the epitope-tagged, mutated receptor showed it maintained the full wild-type biological activity. For extraction of Ste2p, yeast membranes were solubilized with 0.5% n-dodecyl maltoside (DM). Approximately 120 microg of purified alpha-factor receptor was obtained per liter of culture by single-step affinity chromatography using a monoclonal antibody to the rhodopsin epitope. The binding affinity (K(d)) of the purified alpha-factor receptor in DM micelles was 28 nM as compared to K(d)=12.7 nM for Ste2p in cell membranes, and approximately 40% of the purified receptor was correctly folded as judged by ligand saturation binding. About 50% of the receptor sequence was retrieved from MALDI-TOF and nanospray mass spectrometry after CNBr digestion of the purified receptor. The methods described will enable structural studies of the alpha-factor receptor and may provide an efficient technique to purify other GPCRs that have been functionally expressed in yeast.
Authors:
Byung-Kwon Lee; Kyung-Sik Jung; Cagdas Son; Heejung Kim; Nathan C VerBerkmoes; Boris Arshava; Fred Naider; Jeffrey M Becker
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2007-06-20
Journal Detail:
Title:  Protein expression and purification     Volume:  56     ISSN:  1046-5928     ISO Abbreviation:  Protein Expr. Purif.     Publication Date:  2007 Nov 
Date Detail:
Created Date:  2007-10-08     Completed Date:  2008-01-03     Revised Date:  2014-09-22    
Medline Journal Info:
Nlm Unique ID:  9101496     Medline TA:  Protein Expr Purif     Country:  United States    
Other Details:
Languages:  eng     Pagination:  62-71     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Chromatography, Affinity
Molecular Sequence Data
Receptors, Mating Factor / biosynthesis,  isolation & purification*
Saccharomyces cerevisiae / metabolism
Saccharomyces cerevisiae Proteins / biosynthesis,  isolation & purification*
Solubility
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Tandem Mass Spectrometry
Grant Support
ID/Acronym/Agency:
GM 22086/GM/NIGMS NIH HHS; GM 22087/GM/NIGMS NIH HHS; R56 GM022087/GM/NIGMS NIH HHS; R56 GM022087-31A1/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Receptors, Mating Factor; 0/STE2 protein, S cerevisiae; 0/Saccharomyces cerevisiae Proteins
Comments/Corrections

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