RECONSTITUTION OF ACTIVE HUMAN CALCINEURIN FROM RECOMBINANT SUBUNITS EXPRESSED IN BACTERIA

Calcineurin, a protein phosphatase found in eukaryotic cells, presents a challenging problem in heterologous protein expression because it i s both heterodimeric and posttranslationally modified, In this paper, we describe the cloning of both subunits (catalytic A and regulatory B ) of calcineurin from a human cDNA library and their expression at hig h levels in Escherichia coli. The calcineurin A subunit is expressed a s an insoluble glutathione S-transferase fusion protein, while the cal cineurin B subunit is soluble upon direct expression, Catalytically ac tive holoenzyme is derived from the separately expressed subunits usin g a three-step refolding protocol. First, the fusion protein is solubi lized, then it is cleaved at the fusion junction with thrombin, and, f inally, a catalytically competent calcineurin A:calcineurin B:calmodul in complex is reconstituted by cofolding the separately purified compo nents. In addition, we show that a similar refolding protocol can be a pplied to a C-terminally truncated form of calcineurin A, which lacks an autoinhibitory and calmodulin-binding domain. (C) 1995 Academic Pre ss, Inc.