A novel multiple affinity purification tag and its use in identification of proteins associated with a cyclin-CDK complex.
A novel multiple affinity purification (MAFT) or tandem affinity purification (TAP) tag has been constructed. It consists of the calmodulin binding peptide, six histidine residues, and three copies of the hemagglutinin epitope. This 'CHH' MAFT tag allows two or three consecutive purification steps, giving high purity. Active Clb2-Cdc28 kinase complex was purified from yeast cells after inserting the CHH tag into Clb2. Associated proteins were identified using mass spectrometry. These included the known associated proteins Cdc28, Sic1 and Cks1. Several other proteins were found including the 70 kDa chaperone, Ssa1.
Honey
S
S
Cold Spring Harbor Laboratories, Cold Spring Harbor, NY 11724, USA.
Schneider
B L
BL
Schieltz
D M
DM
Yates
J R
JR
Futcher
B
B
D048868:Adaptor Proteins, Signal Transducing; D000251:Adenosine Triphosphatases; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D017489:CDC28 Protein Kinase, S cerevisiae / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins; D002846:Chromatography, Affinity / Q000379:methods; D019926:Cyclin B / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006388:Hemagglutinins / Q000235:genetics / Q000276:immunology; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D009532:Nickel / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
D048868:Adaptor Proteins, Signal Transducing; D000911:Antibodies, Monoclonal; C081185:CKS1 protein, S cerevisiae; C093749:CLB2 protein, S cerevisiae; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D018797:Cell Cycle Proteins; D019926:Cyclin B; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; D006388:Hemagglutinins; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; C086926:SIC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D006639:Histidine; D009532:Nickel; D017489:CDC28 Protein Kinase, S cerevisiae; D000251:Adenosine Triphosphatases; C095973:SSA1 protein, S cerevisiae