摘要
目的:构建重组牛肠激酶轻链的基因工程菌,并进行表达和纯化,以获得高纯度和高活性的重组牛肠激酶轻链蛋白。方法:以GenBank公共数据库中的牛肠激酶轻链基因序列(AccessionNo.NM174439)设计引物,利用RT-PCR合成牛肠激酶轻链基因片段,并克隆进pPIC9K载体,同时在基因N端插进6个组氨酸标签,转化毕赤酵母GS115,进行筛选和诱导表达。产物经镍离子螯和层析和Q-SepharoseFF柱纯化,并酶切融合蛋白检测其活性。结果:培养液中重组牛肠激酶轻链蛋白表达量为3.0mg/L。对含有肠激酶酶切位点的IL-11/MBP融合蛋白进行酶切,结果表明,酶解率可达到90%以上。结论:表达并获得了高纯度的重组肠激酶轻链蛋白,为大规模生产打下了基础。
Objective： To obtain a high-purity and high-activity recombinant bovine enterokinase catalytic subunit protein by inducing the recombinant GS115 yeast containing the bovine enterikinase catalytic subunit gene. Methods： The DNA sequence encoding the light chain （catalytic subunit） of bovine enterokinase （GenBank Accession No. NM174439） was synthesized from the fresh bovine duodenal mucosa by reverse trscriptional polymerase chain reaction and cloned into plas- mid pPIC9K downstream to the gene of fusion partner His-tag. The recombinant pPIC9K-bovine enterokinase light chain vector（pPIC9K-EKL） was expressed in Pichia pastoris GSll5 strain in secreted form. The active recombinant bovine enterokinase light chain （rEKL） was purified on nickel chelating chromatogram using His Binding Resin and Q-Sepharose FF column chromatography. The purified intact rEKL was measured by cleavage fusion protein for its biological activity. Resets： 3.0 mg/L of recombinant bovine enterokinase catalytic subunit protein was obtained from flask culture, and the percentage of cleaved IL-11/MBP fusion protein with enterokinase recognition site was more than 90%. Conclusion： The high-purity bovine recombinant enterokinase catalytic subunit protein was obtained which would be able to lay foundation for its further application of products on a large scale.
出处
《生物技术通讯》
CAS
2006年第1期5-8,共4页
Letters in Biotechnology
基金
国家自然科学基金项目(30471250)
关键词
重组牛肠激酶
毕赤酵母
分泌表达
活性分析
recombinant bovine enterokinase
secreted expression
fusion protein
activity assay
