Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method

Field, J., Nikawa, J., Broek, D., MacDonald, B., Rodgers, L., Wilson, I. A., Lerner, R. A., Wigler, M. H. (May 1988) Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Molecular & Cellular Biology, 8 (5). pp. 2159-65. ISSN 0270-7306

URL: https://www.ncbi.nlm.nih.gov/pubmed/2455217
DOI: 10.1128/MCB.8.5.2159

Abstract

We developed a method for immunoaffinity purification of Saccharomyces cerevisiae adenylyl cyclase based on creating a fusion with a small peptide epitope. Using oligonucleotide technology to encode the peptide epitope we constructed a plasmid that expressed the fusion protein from the S. cerevisiae alcohol dehydrogenase promoter ADH1. A monoclonal antibody previously raised against the peptide was used to purify adenylyl cyclase by affinity chromatography. The purified enzyme appeared to be a multisubunit complex consisting of the 200-kilodalton adenylyl cyclase fusion protein and an unidentified 70-kilodalton protein. The purified protein could be activated by RAS proteins. Activation had an absolute requirement for a guanine nucleoside triphosphate.

Item Type: Paper
Additional Information:
Uncontrolled Keywords: Adenylate Cyclase isolation & purification Amino Acid Sequence Antibodies Monoclonal immunology Chromatography Affinity Epitopes immunology Fungal Proteins isolation & purification physiology Guanine Nucleotides pharmacology Molecular Sequence Data Peptides immunology Recombinant Fusion Proteins immunology isolation & purification Saccharomyces cerevisiae enzymology ras Proteins
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > genes, structure and function > genes: types > RAS
organism description > yeast > Saccharomyces
biotechnology > chromatography > affinity chromatography
biotechnology > chromatography
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > genes, structure and function > genes: types > oncogene
CSHL Authors:
Communities: CSHL labs > Wigler lab
Depositing User: CSHL Librarian
Date: May 1988
Date Deposited: 13 Apr 2012 20:07
Last Modified: 04 Nov 2016 20:46
PMCID: PMC363397
Related URLs:
URI: http://repository.cshl.edu/id/eprint/26181

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