Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method.
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.
Field
J
J
Cold Spring Harbor Laboratory, New York 11724.
Nikawa
J
J
Broek
D
D
MacDonald
B
B
Rodgers
L
L
Wilson
I A
IA
Lerner
R A
RA
Wigler
M
M
D000262:Adenylyl Cyclases / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D002846:Chromatography, Affinity; D000939:Epitopes / Q000276:immunology; D005656:Fungal Proteins / Q000302:isolation & purification* / Q000502:physiology*; D006150:Guanine Nucleotides / Q000494:pharmacology; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D018631:ras Proteins
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005656:Fungal Proteins; D006150:Guanine Nucleotides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D018631:ras Proteins; D000262:Adenylyl Cyclases