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 Ca2+-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.
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|Published - 1992