Affinity purification and characterization of a G-protein coupled receptor, Saccharomyces cerevisiae Ste2p

Lee, Byung-Kwon
Jung, Kyung-Sik
Son, Çağdaş Devrim
Kini, Heejung
VerBerknioes, Nathan C.
Arshava, Boris
Naider, Fred
Becker, Jeffrey M.
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 a-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 solubilize with 0.5% n-dodecyl maltoside (DM). Approximately 120 mu g of purified a-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 a-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 a-factor receptor and may provide an efficient technique to purify other GPCRs that have been functionally expressed in yeast. (c) 2007 Elsevier Inc. All rights reserved.


Purification, characterization, crystallization and preliminary x-ray structure determination of scytalidium thermophilum bifunctional catalase and identification of its catechol oxidase activity
Sutay, Didem; Bakır, Ufuk; Department of Chemical Engineering (2007)
In this study, the aim was identification and classification of the enzyme having phenol oxidase activity produced by a thermophilic fungus, Scytalidium thermophilum. For this purpose, enzyme production, purification, biochemical characterization and structural analysis by X-ray crystallography studies have been performed. At the beginning of the research, this enzyme was considered as a phenol oxidase and analyzed accordingly. However, during purification, amino acid sequencing and structural studies, the ...
Kinetic analyses of the effects of temperature and light intensity on growth, hydrogenm production and organic acid utilization by rhodobacter capsulatus
Sevinç, Pelin; Gündüz, Ufuk; Department of Biotechnology (2010)
Effects of temperature and light intensity on photofermentative hydrogen production by Rhodobacter capsulatus DSM1710 by use of acetic and lactic acids as substrates were studied. Experiments were conducted at 20, 30 and 38oC incubator temperatures under light intensities in the 1500 – 7000 lux range. pH of the medium and quantity of hydrogen forming together with quantity of biomass, and concentrations of acetic, lactic, formic, butyric and propionic acids in the medium were determined periodically. Growth...
Influence of oxygen transfer on benzaldehyde lyase production by recombinant Escherichia coli BL21(DE3) pLySs
Angardi, Vahideh; Çalık, Pınar; Department of Chemical Engineering (2007)
In this study, the effects of oxygen transfer conditions on the synthesis of the enzyme benzaldehyde lyase as intracellular in recombinant E. coli BL21 (DE3) pLysS was investigated sistematically and a comprehensive model was developed to determine benzaldehyde lyase activity. For this purpose, the research program was carried out in mainly two parts. In the first part of study, the effects of oxygen transfer together with the mass transfer coefficient (KLa), enhancement factor E (=KLa/KLao), volumetric oxy...
Molecular cloning and co-expression of Thermoplasma volcanium proteasome subunit genes
Kocabıyık, Semra; Zwickl, Peter; Ozdogan, Seda (Elsevier BV, 2010-10-01)
In this study we describe, the construction of a co-expression vector allowing simultaneous production of Thermoplasma volcanium 20S proteasome alpha- and beta-subunits in Escherichia coli. This heterologous expression system provided high level production of fully active 205 proteasome that can be purified easily by using a conventional two-step chromatographic technique. The recombinant proteasome was purified to homogeneity 12-fold with a specific activity of 26.5 U/mg. Sodium dodecyl sulfate-polyacrylam...
Production of recombinant proteins by yeast cells
Celik, Eda; Çalık, Pınar (Elsevier BV, 2012-09-01)
Yeasts are widely used in production of recombinant proteins of medical or industrial interest. For each individual product, the most suitable expression system has to be identified and optimized, both on the genetic and fermentative level, by taking into account the properties of the product, the organism and the expression cassette. There is a wide range of important yeast expression hosts including the species Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Kluyveromyces lactis, Schizosa...
Citation Formats
B.-K. Lee et al., “Affinity purification and characterization of a G-protein coupled receptor, Saccharomyces cerevisiae Ste2p,” PROTEIN EXPRESSION AND PURIFICATION, pp. 62–71, 2007, Accessed: 00, 2020. [Online]. Available: