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Title: Affinity Purification and Characterization of a G-Protein Coupled Receptor, Saccharomyces cerevisiae Ste2p

Abstract

We present a rare example of a biologically active G protein coupled receptor (GPCR) whose purity and identity were verified by mass spectrometry after being purified to near homogeneity from its native system. An overexpression vector was constructed to encode the Saccharomyces cerevisiae GPCR -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. Tests of the epitope-tagged, mutated receptor showed it maintained its full biological activity. For extraction of Ste2p, yeast membranes were solubilized with 0.5 % n-dodecyl maltoside (DM). Approximately 120 g of purified -factor receptor was obtained per liter of culture by single-step affinity chromatography using a monoclonal antibody to the rhodopsin epitope. The binding affinity (Kd) of the purified -factor receptor in DM micelles was 28 nM as compared to Kd = 12.7 nM for Ste2p in cell membranes, and approximately 40 % of the purified receptor was correctly folded as judged by ligandmore » saturation binding. About 50 % of the receptor sequence was retrieved from MALDITOF and nanospray mass spectrometry after CNBr digestion of the purified receptor. The methods described will enable structural studies of the -factor receptor and may provide an efficient technique to purify other GPCRs that have been functionally expressed in yeast.« less

Authors:
 [1];  [1];  [2];  [1];  [2];  [3];  [3];  [2]
  1. University of Tennessee, Knoxville (UTK)
  2. ORNL
  3. College of Staten Island
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
930940
DOE Contract Number:  
DE-AC05-00OR22725
Resource Type:
Journal Article
Resource Relation:
Journal Name: Protein Expression and Purification; Journal Volume: 56; Journal Issue: 1
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; AFFINITY; CELL MEMBRANES; MASS SPECTROSCOPY; PROTEINS; PURIFICATION; SACCHAROMYCES CEREVISIAE

Citation Formats

Lee, Byung-Kwon, Jung, Kyung-Sik, Son, Cagdas D, Kim, Heejung, Verberkmoes, Nathan C, Arshava, Boris, Naider, Fred, and Becker, Jeffrey Marvin. Affinity Purification and Characterization of a G-Protein Coupled Receptor, Saccharomyces cerevisiae Ste2p. United States: N. p., 2007. Web. doi:10.1016/j.pep.2007.06.002.
Lee, Byung-Kwon, Jung, Kyung-Sik, Son, Cagdas D, Kim, Heejung, Verberkmoes, Nathan C, Arshava, Boris, Naider, Fred, & Becker, Jeffrey Marvin. Affinity Purification and Characterization of a G-Protein Coupled Receptor, Saccharomyces cerevisiae Ste2p. United States. doi:10.1016/j.pep.2007.06.002.
Lee, Byung-Kwon, Jung, Kyung-Sik, Son, Cagdas D, Kim, Heejung, Verberkmoes, Nathan C, Arshava, Boris, Naider, Fred, and Becker, Jeffrey Marvin. Mon . "Affinity Purification and Characterization of a G-Protein Coupled Receptor, Saccharomyces cerevisiae Ste2p". United States. doi:10.1016/j.pep.2007.06.002.
@article{osti_930940,
title = {Affinity Purification and Characterization of a G-Protein Coupled Receptor, Saccharomyces cerevisiae Ste2p},
author = {Lee, Byung-Kwon and Jung, Kyung-Sik and Son, Cagdas D and Kim, Heejung and Verberkmoes, Nathan C and Arshava, Boris and Naider, Fred and Becker, Jeffrey Marvin},
abstractNote = {We present a rare example of a biologically active G protein coupled receptor (GPCR) whose purity and identity were verified by mass spectrometry after being purified to near homogeneity from its native system. An overexpression vector was constructed to encode the Saccharomyces cerevisiae GPCR -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. Tests of the epitope-tagged, mutated receptor showed it maintained its full biological activity. For extraction of Ste2p, yeast membranes were solubilized with 0.5 % n-dodecyl maltoside (DM). Approximately 120 g of purified -factor receptor was obtained per liter of culture by single-step affinity chromatography using a monoclonal antibody to the rhodopsin epitope. The binding affinity (Kd) of the purified -factor receptor in DM micelles was 28 nM as compared to Kd = 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 MALDITOF and nanospray mass spectrometry after CNBr digestion of the purified receptor. The methods described will enable structural studies of the -factor receptor and may provide an efficient technique to purify other GPCRs that have been functionally expressed in yeast.},
doi = {10.1016/j.pep.2007.06.002},
journal = {Protein Expression and Purification},
number = 1,
volume = 56,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}