Protein A-calmodulin fusions: a novel approach for investigating calmodulin function in yeast.
A novel gene fusion approach which may be of more general use has been developed for investigating the function of calmodulin in the budding yeast Saccharomyces cerevisiae. By fusing a portion of the Staphylococcus aureus spa gene (encoding protein A) to CMD1, the S. cerevisiae gene encoding calmodulin, we have generated a yeast calmodulin with an affinity tag able to bind immunoglobulins. The chimaeric protein A-calmodulin (ProtA-CaM) polypeptide functions in vivo and shows Ca(2+)-dependent binding to calmodulin target proteins. The spa-CMD1 fusion has been used (i) to prepare (by affinity chromatography) a fraction of yeast proteins which interact with calmodulin, (ii) to isolate genes encoding calmodulin target proteins by direct screening of an expression library, and (iii) to visualize calmodulin-binding proteins in crude extracts by Western blot analysis.
Stirling
D A
DA
Department of Biochemistry, University of Dundee, UK.
Petrie
A
A
Pulford
D J
DJ
Paterson
D T
DT
Stark
M J
MJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002147:Calmodulin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D015723:Gene Library; D000072235:Mating Factor; D008969:Molecular Sequence Data; D010455:Peptides / Q000494:pharmacology; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013205:Staphylococcal Protein A / Q000235:genetics* / Q000378:metabolism
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D004274:DNA, Recombinant; D010455:Peptides; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D000072235:Mating Factor