Expression and purification of recombinant human granzyme B from Pichia pastoris

Granzyme B is a cytotoxic lymphocyte granule serine proteinase that is pivo tal in the induction of target cell apoptosis. Here we describe the express ion of recombinant human granzyme B in Pichia pastoris as a chimeric zymoge n comprising the cu-factor signal sequence, a prodomain including an entero kinase cleavage site, and the mature granzyme B sequence followed by a hexa histidine tag, Inactive zymogen is purified from the medium by immobilized cobalt chelate affinity chromatography and then activated by enterokinase ( final yield is approximately 1 mg per liter). The recombinant enzyme resemb les native granzyme B in size and glycosylation, hydrolyzes the substrate B oc-Ala-Ala-Asp-thiobenzyl ester with equivalent efficiency (K-m 82 mu M; k( cat) 12 s(-1)), processes procaspase-3 to subunit form, and is inhibited by the cognate serpin PI-9. It efficiently induces DNA degradation and apopto sis of human cells. The availability of recombinant human granzyme B will f acilitate further investigation of its structure and role in immune effecto r cells. (C) 1999 Academic Press.