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. 1988 May;8(5):2159–2165. doi: 10.1128/mcb.8.5.2159

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

J Field 1, J Nikawa 1, D Broek 1, B MacDonald 1, L Rodgers 1, I A Wilson 1, R A Lerner 1, M Wigler 1
PMCID: PMC363397  PMID: 2455217

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.

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Selected References

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