Expression, purification, and characterization of human enteropeptidase catalytic subunit in Escherichia coli
Expression, purification, and characterization of human enteropeptidase catalytic subunit in Escherichia coli
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DOI：
10.1016/s1046-5928(03)00159-1
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发表时间：
2003-09-01
影响因子：
1.6
通讯作者：
Kirpichnikov, MP
中科院分区：
文献类型：
--
作者：
Gasparian, ME;Ostapchenko, VG;Kirpichnikov, MP
肠肽酶（同义词：肠激酶，EC 3.4.21.9）是一种肠刷状缘的异二聚体丝氨酸蛋白酶，通过高度特异性切割序列（ASP）（4）-Lys后的胰蛋白酶原激活肽来激活胰蛋白酶原。通过聚合酶链反应从26个寡核苷酸合成编码人肠肽酶（GenBank登录号U 09860）的轻链（催化亚基）的DNA序列，并将其克隆到质粒pET-32a中融合伴侣硫氧还蛋白基因的下游，紧接在编码肠肽酶识别位点的DNA序列之后。硫氧还蛋白/人肠肽酶轻链融合蛋白在大肠杆菌BL 21（DE 3）中以可溶性和不溶性两种形式表达。可溶性重组融合蛋白不能进行自催化切割和活化，但通过溶解和复性融合蛋白，实现了重组人肠肽酶轻链（L-HEP）的自催化切割和活化，并在琼脂糖连接的大豆胰蛋白酶抑制剂上纯化了活性L-HEP。纯化的L-HEP裂解合成肽底物Gly-Asp-Asp-Asp-Asp-Lys-β-萘酰胺，动力学参数为K-m = 0.16 mM和k（cat）= 115 s（-1），裂解小酯Z-Lys-SBzl，K-m = 140 μ M，k（cat）= 133 s（-1）。L-HEP与大豆胰蛋白酶抑制剂缓慢结合，并且小酯Z-Lys-SBzl裂解被抑制，Ki（*）= 2.3nM。在相同条件下，L-HEP消化的硫氧还蛋白/人表皮生长因子融合蛋白比相同活性单位的牛重组轻链（EKMax，Invitrogen）快五倍。(C)2003 Elsevier Science（美国）。All rights reserved.
Enteropeptidase (synonym: enterokinase, EC 3.4.21.9) is a heterodimeric serine protease of the intestinal brush border that activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the sequence (ASP)(4)-Lys. The DNA sequence encoding the light chain (catalytic subunit) of human enteropeptidase (GenBank Accession No. U09860) was synthesized from 26 oligonucleotides by polymerase chain reaction and cloned into plasmid pET-32a downstream to the gene of fusion partner thioredoxin immediately after the DNA sequence encoding enteropeptidase recognition site. The fusion protein thioredoxin/human enteropeptidase light chain was expressed in Escherichia coli BL21(DE3) strain in both soluble and insoluble forms. The soluble recombinant fusion protein failed to undergo autocatalytic cleavage and activation; however, autocatalytic cleavage and activation of recombinant human enteropeptidase light chain (L-HEP) were achieved by solubilization and renaturation of the fusion protein from inclusion bodies and the active L-HEP was purified on agarose-linked soybean trypsin inhibitor. The purified L-HEP cleaved the synthetic peptide substrate Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide with kinetic parameters K-m = 0.16 mM and k(cat) = 115 s(-1) and small ester Z-Lys-SBzl with K-m = 140 muM, k(cat) = 133 s(-1). L-HEP associated with soybean trypsin inhibitor slowly and small ester Z-Lys-SBzl cleavage was inhibited with K-i(*) = 2.3 nM. L-HEP digested thioredoxin/human epidermal growth factor fusion protein five times faster than equal activity units of bovine recombinant light chain (EKMax, Invitrogen) at the same conditions. (C) 2003 Elsevier Science (USA). All rights reserved.