[目的]在原核表达系统中实现人肠激酶轻链(Human enterokinase light chain, hEKL)的表达和纯化.[方法]通过PCR扩增得到编码hEKL的基因片段,利用基因重组技术构建原核表达质粒pMAL-s-hEKL,在Escherichia coli中进行诱导表达,菌体经超声破碎后利用Amylose亲和柱对目标蛋白进行纯化,并利用Tricine SDS-PAGE检测酶的切割活性.[结果]目的基因能够以可溶形式表达,每升发酵液可纯化得到40 mg纯度在97％以上的MBP-hEKL蛋白,活性检测表明该酶可以对含有肠激酶识别序列的蛋白进行特异性切割,酶活力达到6.0× 105 U/μmol.%[Objective] To express and purify human enterokinase light chain (HEKL) in pro-karyotic expression system and detecting its activity in vitro.[Methods] The fragment of hEKL gene was amplified by PCR and cloned into plasmid pMAL-s downstream to the gene of fusion partner MBP-tag.The recombinant plasmid pMAL-s-hEKL was transformed into E.coli BL21(DE3).After induced expression, affinity chromatography was amplified to purify the target protein and Tricine SDS-PAGE was used to analyze the enzymatic activity.[Results] Majority of the fusion protein MBP-hEKL was expressed in soluble form.40 mg protein was obtained with a purity of more than 97% from 1 L fermentation broth.Activity analysis showed that MBP-hEKL could cleavage the fusion protein with enterokinase recognition site specially and the specific activity was reached to 6.0×105 U/μmol.
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