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

Mol Cell Biol. 1988 May;8(5):2159-65. doi: 10.1128/mcb.8.5.2159-2165.1988.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenylyl Cyclases / isolation & purification*
  • Amino Acid Sequence
  • Antibodies, Monoclonal / immunology
  • Chromatography, Affinity
  • Epitopes / immunology
  • Fungal Proteins / isolation & purification*
  • Fungal Proteins / physiology*
  • Guanine Nucleotides / pharmacology
  • Molecular Sequence Data
  • Peptides / immunology
  • Recombinant Fusion Proteins / immunology
  • Recombinant Fusion Proteins / isolation & purification
  • Saccharomyces cerevisiae / enzymology*
  • ras Proteins*

Substances

  • Antibodies, Monoclonal
  • Epitopes
  • Fungal Proteins
  • Guanine Nucleotides
  • Peptides
  • Recombinant Fusion Proteins
  • ras Proteins
  • Adenylyl Cyclases