Huang Lei, Ruan Hong, Gu Weiyan, Xu Zhinan, Cen Peilin, Fan Limei
Institute of Bioengineering, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou, PR China.
Prep Biochem Biotechnol. 2007;37(3):205-17. doi: 10.1080/10826060701386695.
Enterokinase (EC 3.4.21.9) is a serine proteinase of the intestinal brush border that exhibits specificity for the sequence (Asp)(4)-Lys and converts trypsinogen into its active form, trypsin. A codon optimized sequence coding light chain (catalytic subunit) of bovine enterokinase gene (sBEKLC) was synthesized, and it was fused with DsbA to construct the expression vector (pET39-sBEKLC). Then, the plasmid was transformed into E. coli BL21 (DE3) for expression. Under optimal conditions, the volumetric productivity of fusion protein reached 151.2 mg L(-1), i.e., 80.6 mg sBEKLC L(-1). The cold osmotic shock technique was successfully used to extract sBEKLC from periplasmic space, and nickel affinity chromatography was employed to obtain mature sBEKLC. Finally, about 6.8 mg of bioactive sBEKLC was purified from 1 liter fermentation broth and could be used to cleave one tested fusion protein with an inter-domain enteropeptidase recognition site. This work will be helpful for large-scale production of this increasingly demanded enterokinase.
肠激酶（EC 3.4.21.9）是一种存在于小肠刷状缘的丝氨酸蛋白酶，对（天冬氨酸）4-赖氨酸序列具有特异性，并能将胰蛋白酶原转化为其活性形式——胰蛋白酶。合成了编码牛肠激酶基因轻链（催化亚基）的密码子优化序列（sBEKLC），并将其与DsbA融合构建表达载体（pET39-sBEKLC）。然后，将该质粒转化到大肠杆菌BL21（DE3）中进行表达。在最佳条件下，融合蛋白的体积产率达到151.2 mg L-1，即80.6 mg sBEKLC L-1。成功地利用冷渗透休克技术从周质空间提取sBEKLC，并采用镍亲和层析法获得成熟的sBEKLC。最后，从1升发酵液中纯化得到约6.8 mg的生物活性sBEKLC，可用于切割一种具有结构域间肠肽酶识别位点的测试融合蛋白。这项工作将有助于大规模生产这种需求日益增长的肠激酶。
