IN-VIVO CALCINEURIN CRYSTALS FORMED USING THE BACULOVIRUS EXPRESSION SYSTEM

Calcineurin is a heterodimeric phosphatase involved in the signal tran sduction of antigen-activated T cells. Coexpression of its two subunit s, the regulatory subunit from human and the catalytic subunit from Ne urospora crassa in cultured insect cells using the baculovirus express ion system results in the formation of very large crystals in the cyto plasm. The crystals are formed initially in vesicles, but their subseq uent growth appears to be uninhibited and continues without the need o f an enclosing membrane until the host cell lyses. Although these in v ivo crystals are low in population, ranging only 0-3 per cell, they ar e extremely large, over 10 mu m in some cases. Biochemical assays conf irm their calcineurin origin, with the regulatory subunit incorporated being myristoylated, although both the myristoylated and unmyristoyla ted forms are expressed. The lattice structure of the in vivo crystals , with a spacing of 5.5 nm, is preserved with the regular electron mic roscopic (EM) specimen preparation procedure. (C) 1996 Wiley-Liss, Inc .