Azhar Mahammad, Somashekhar R
a Jawaharlal Nehru Technological University, Department of Biotechnology , Hyderabad , India.
Prep Biochem Biotechnol. 2015;45(3):268-78. doi: 10.1080/10826068.2014.907185.
Enteropeptidase (EC 3.4.21.9) is the glycoprotein enzyme in the small intestine that triggers the activation of the zymogens in pancreatic juice by converting trypsinogen into trypsin. Because of its physiological significance, there have been many studies on the expression, purification, and characterization of enteropeptidase from different species. The baculovirus expression system has been commonly used in research communities and scientific industries for the production of high levels of recombinant proteins, which require posttranslational modifications for functional activity. In the present study, we isolated bovine enteropeptidase catalytic subunit gene from Bos taurus indicus (GenBank accession no. KC756844), and cloned it in pFast Bac HT "A" baculovirus expression donor vector, under the polyhedrin promoter. Recombinant bovine enteropeptidase was expressed in SF-9 insect cells with high expression levels. Recombinant enteropeptidase was purified using Ni-NTA affinity chromatography. A 6-mg quantity of pure active protein was obtained from 100 mL culture using this approach. Its activity and kinetic parameters were determined by cleavage of its fluorogenic substrate Gly-(Asp) 4-Lys-β-naphthylamide. The recombinant bovine enteropeptidase showed a K m value of 0.75 ± 0.02 mM with K cat 25 ± 1 s.
肠肽酶（EC 3.4.21.9）是小肠中的一种糖蛋白酶，它通过将胰蛋白酶原转化为胰蛋白酶来触发胰液中酶原的激活。由于其生理意义，已经有许多关于不同物种肠肽酶的表达、纯化和特性的研究。杆状病毒表达系统已在研究团体和科学产业中普遍用于生产高水平的重组蛋白，这些重组蛋白需要翻译后修饰才能具有功能活性。在本研究中，我们从印度瘤牛（GenBank登录号KC756844）中分离出牛肠肽酶催化亚基基因，并将其克隆到pFast Bac HT“A”杆状病毒表达供体载体中，置于多角体蛋白启动子之下。重组牛肠肽酶在SF-9昆虫细胞中高表达。重组肠肽酶通过镍-氮三乙酸亲和层析进行纯化。用这种方法从100 mL培养物中获得了6 mg纯活性蛋白。通过切割其荧光底物甘氨酰-（天冬氨酸）4-赖氨酸-β-萘酰胺来测定其活性和动力学参数。重组牛肠肽酶的K m值为0.75±0.02 mM，K cat为25±1 s-1。
