Author:
Field J,Nikawa J,Broek D,MacDonald B,Rodgers L,Wilson I A,Lerner R A,Wigler M
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.
Publisher
American Society for Microbiology
Subject
Cell Biology,Molecular Biology
Cited by
868 articles.
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