重组人丝氨酸蛋白酶抑制因子Hespintor Kazal结构域的原核表达、纯化及活性鉴定

Prokaryotic expression, purification and activity analysis of recombinant human serine protease inhibitor Hespintor Kazal Domain

Hespintor是应用抑制消减杂交技术(SSH)从肝母细胞瘤细胞系HepG2中筛选得到的一未知功能蛋白,序列分析表明该蛋白属于Kazal型丝氨酸蛋白酶抑制因子(Serine proteinase inhibitor,Serpin)家族中的一个分泌型新成员,具有与食管癌相关基因2(Esophageal cancer related gene 2,ECRG2)高度同源的Serpin基本结构。为了进一步阐明Hespintor的生物学功能,必须得到纯化的Hespintor蛋白。先将Hespintor Kazal结构域编码序列亚克隆至原核表达载体pET-40b(+),转化至Rosetta(DE3)表达宿主菌中。经0.25 mmol/L IPTG,30℃诱导5 h获得了分子量约为42 kDa的Hespintor-Kazal重组融合蛋白的优化表达,Western blotting证实了重组蛋白的特异性。Hespintor-Kazal重组融合蛋白以包涵体形式在宿主菌中表达,利用金属螯合亲和层析和阴离子交换层析柱对重组蛋白进行两步纯化。初步的活性鉴定表明,纯化的Hespintor-Kazal重组融合蛋白能特异性抑制胰蛋白酶的水解活性,提示Hespintor具有作为一种新型抗肿瘤药物的潜在开发价值。

Hespintor is an unknown function protein that was got from hepatoblastoma cell lines HepG2 by suppression subtractive hybridization technique （SSH）, sequence analysis showed that the protein is a new member of secretory type of Kazal type serine protease inhibitor （Serpin） family, and has high homology with esophageal cancer related gene 2 （ECRG2）. The coding sequence of Hespintor＇s Kazal domain was subcloned into prokaryotic expression vector pET-40b（＋）, then transformed into Rosetta （DE3）. A recombinant protein about 42 kDa in the form of inclusion body was optimization expressed by inducing with 0.25 mmol/L IPTG, 30 ~C for 5 h. and its specificity was confirmed via Western blotting. The recombinant protein was purified by metal chelate affinity chromatography （MCAC） and anion-exchange chromatography. The preliminary experimental result showed that the recombinant protein can inhibit trysin hydrolysis activity specifically. The result clearly demonstrated that Hespintor, as a novel member of Serpin, would be valuable in developing anti-tumor agents.