Sun J, Bird C H, Buzza M S, McKee K E, Whisstock J C, Bird P I
Monash Medical School, Box Hill Hospital, Australia.
Biochem Biophys Res Commun. 1999 Aug 2;261(2):251-5. doi: 10.1006/bbrc.1999.0989.
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.
颗粒酶B是一种细胞毒性淋巴细胞颗粒丝氨酸蛋白酶，在诱导靶细胞凋亡中起关键作用。在此，我们描述了重组人颗粒酶B在毕赤酵母中的表达，它是一种嵌合酶原，包含α-因子信号序列、一个含有肠激酶切割位点的前结构域、成熟的颗粒酶B序列以及一个六组氨酸标签。无活性的酶原通过固定化钴螯合亲和层析从培养基中纯化出来，然后用肠激酶激活（最终产量约为每升1毫克）。重组酶在大小和糖基化方面类似于天然颗粒酶B，以相同效率水解底物Boc-Ala-Ala-Asp-硫代苄酯（K(m) 82微摩尔；k(cat) 12秒-1），将procaspase-3加工成亚基形式，并被同源丝氨酸蛋白酶抑制剂PI-9抑制。它能有效诱导人细胞的DNA降解和凋亡。重组人颗粒酶B的可得性将有助于进一步研究其在免疫效应细胞中的结构和作用。
