title: Expression and purification of recombinant human granzyme B from Pichia pastoris.
abstract: Granzyme B is a cytotoxic lymphocyte granule serine proteinase that is pivotal in the induction of target cell apoptosis. Here we describe the expression of recombinant human granzyme B in Pichia pastoris as a chimeric zymogen comprising the alpha-factor signal sequence, a prodomain including an enterokinase cleavage site, and the mature granzyme B sequence followed by a hexahistidine 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 resembles native granzyme B in size and glycosylation, hydrolyzes the substrate Boc-Ala-Ala-Asp-thiobenzyl ester with equivalent efficiency (K(m) 82 microM; 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 apoptosis of human cells. The availability of recombinant human granzyme B will facilitate further investigation of its structure and role in immune effector cells.
authors.affiliation: Monash Medical School, Box Hill Hospital, Australia.
authors.forename: J
authors.initials: J
authors.lastname: Sun
authors.forename: C H
authors.initials: CH
authors.lastname: Bird
authors.forename: M S
authors.initials: MS
authors.lastname: Buzza
authors.forename: K E
authors.initials: KE
authors.lastname: McKee
authors.forename: J C
authors.initials: JC
authors.lastname: Whisstock
authors.forename: P I
authors.initials: PI
authors.lastname: Bird
mesh_terms: D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects; D001483:Base Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D053804:Granzymes; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases