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Protein A–calmodulin fusions: a novel approach for investigating calmodulin function in yeast
Author(s) -
Stirling Douglas A.,
Petrie Alison,
Pulford David J.,
Paterson David T. W.,
Stark Michael J. R.
Publication year - 1992
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1992.tb01519.x
Subject(s) - calmodulin , calmodulin binding proteins , biology , saccharomyces cerevisiae , fusion protein , yeast , gene , biochemistry , tandem affinity purification , function (biology) , binding protein , affinity chromatography , fusion gene , microbiology and biotechnology , recombinant dna , enzyme
Summary 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.