Abstract
Publisher Summary The chapter describes the purification and functional assay of yFKBP12 from Saccharomyces cerevisiae (S. cerevisiae) and the expression and purification of recombinant yFKBP12. FKBP12 can be assayed in three ways: by peptidylprolyl isomerase (PPIase) activity, by direct binding of the immunosuppressive ligand FK506, and by inhibition of calcineurin phosphatase activity by FK506 yFKBPI2 complexes. The chapter describes purification of FKBP 12 from S. cerevisiae, expression and purification of recombinant yeast FKBP12, peptidylprolyl isomerase activity of yeast FKBP12 and FKBP13, and calcineurin phosphatase inhibition by yFKBP 12 drug complex. The immunophilins constitute a class of ubiquitously expressed proteins named for their ability to bind the immunosuppressive drugs cyclosporin A (CsA), FK506 (Prograf), and rapamycin (RAPA). The genes for five CsA-binding proteins and three FK506/RAPA-binding proteins have so far been characterized in S. cerevisiae . The protein products of these genes possess properties essentially identical to those of the mammalian equivalents and bind either CsA or FK506/RAPA with high affinity and possess peptidylprolyl cis-trans -isomerase (PPIase, EC 5.2.1.8) activity.
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