Cloning and functional expression of a cDNA encoding the catalytic subunit of bovine enterokinase.
Cloning and functional expression of a cDNA encoding the catalytic subunit of bovine enterokinase.
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DOI：
10.1016/s0021-9258(19)49464-7
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发表时间：
1993-11
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影响因子：
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通讯作者：
E. Lavallie;A. Rehemtulla;Lisa A. Racie;E. Diblasio;C. Ferenz;K. L. Grant;A. Light;John;McCoy
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文献类型：
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作者：
E. Lavallie;A. Rehemtulla;Lisa A. Racie;E. Diblasio;C. Ferenz;K. L. Grant;A. Light;John;McCoy
肠激酶（肠肽酶）是一种异二聚体丝氨酸蛋白酶，通过对 (Asp)4-Lys 序列后的胰蛋白酶原激活肽进行高度特异性切割，负责胰蛋白酶原的生理激活。在本文中，我们报道了编码牛肠激酶催化结构域（轻链）的 cDNA 的克隆和功能表达。该克隆 cDNA 的核苷酸序列预测了 235 个氨基酸的多肽，该多肽与参与消化、凝血和纤溶的多种哺乳动物丝氨酸蛋白酶具有高度同源性。我们开发了一种新的酶表达方法，该方法利用哺乳动物丝氨酸蛋白酶 PACE 的分泌前导序列和前肽与肠激酶轻链氨基末端融合。通过与人 PACE 或酵母 KEX2 共表达，实现了配对二元氨基酸裂解酶 (PACE) 前肽的有效裂解。成熟产物在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上的迁移量为 43,000 Da，与牛十二指肠衍生的轻链相当，并且在裂解肠激酶特异性荧光底物 Gly-(Asp)4-Lys-β-萘酰胺方面表现出高水平的活性。重组单链形式的肠激酶也能够激活胰蛋白酶原，这表明即使在不存在非催化性肠激酶重链的情况下，该酶对其天然底物的特异性也得以保留。
Enterokinase (enteropeptidase) is a heterodimeric serine protease that is responsible for the physiological activation of trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the sequence (Asp)4-Lys. In this paper, we report the cloning and functional expression of a cDNA encoding the catalytic domain (light chain) of bovine enterokinase. The nucleotide sequence of this cloned cDNA predicts a 235-amino acid polypeptide that shares a high degree of homology with a variety of mammalian serine proteases involved in digestion, coagulation, and fibrinolysis. We have developed a novel expression method for the enzyme which utilizes the secretory leader and propeptide of the mammalian serine protease PACE fused to the enterokinase light chain amino terminus. Efficient cleavage of the paired dibasic amino acid cleaving enzyme (PACE) propeptide was achieved by coexpression with human PACE or yeast KEX2. The mature product migrates at 43,000 Da on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, comparable to light chain derived from bovine duodena, and exhibited high levels of activity in cleaving the enterokinase-specific fluorogenic substrate Gly-(Asp)4-Lys-beta-naphthylamide. The recombinant single-chain form of enterokinase was also capable of activating trypsinogen, indicating that the specificity of the enzyme for its natural substrate is retained even in the absence of the noncatalytic enterokinase heavy chain.