摘要
肠激酶作为一种丝氨酸蛋白酶,能够识别顺序-Asp-Asp-Asp-Asp-Lys,水解赖氨酸C端的肽键.根据NCBI中牛肠激酶基因序列(L19663)设计18条引物,通过重叠延伸拼接PCR技术(SOE-PCR)扩增两端带有酶切位点的牛肠激酶轻链基因.构建表达载体pET32a-EK,表达载体转化E.coli BL21(DE3),利用IPTG诱导表达嵌合蛋白,并用离子交换柱层析纯化肠激酶,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.
Enterokinase is a serine protease that recognizes Asp-Asp-Asp-Asp-Lys sequence and cleaves the C-terminal peptide bond of the lysine residue.Design eighteen primers according to the sequence of NCBI,slight chain of bovine enterokinase gene was synthesized by SOE-PCR.Construct the expression vector(pET32a-EK).Then the plasmid was transformed into E.coli BL21(DE3)for expression.IPTG was added to express the fusion protein,use lon exchange column to purificy the enterokinase.The purified enterokinase was obtained which would be able to lay foudation for ite further application of products on a large scale.
出处
《辽宁大学学报（自然科学版）》
CAS
2012年第1期12-14,共3页
Journal of Liaoning University：Natural Sciences Edition
关键词
肠激酶
表达
纯化
enterokinase
expression
purification
