血管性血友病因子裂解酶金属蛋白酶域的克隆和表达

目的:克隆和表达血管性血友病因子裂解酶(vWF-cp)的金属蛋白酶域。方法:应用PCR从含有人vWF-cp金属蛋白酶域的质粒中扩增出该区域,克隆至pUC18载体后行序列分析,并将其重组于带有6×HisTag的融合蛋白表达载体pET28a(+)中,在大肠杆菌DE3／plySs中诱导表达。融合蛋白经Ni-NTA柱纯化后,采用Westernblot印迹鉴定。结果:PCR扩增得到658bp的vWF-cp金属蛋白酶域的基因片段,测序结果与GenBank序列一致。重组表达质粒经异丙基硫代-β-D-半乳糖苷(IPTG)诱导5h后表达的融合蛋白占菌体总蛋白的28％,进一步纯化后其纯度在95％以上。结论:在大肠杆菌中高效表达了人vWF-cp的金属蛋白酶结构域,为深入研究vWF-cp结构与功能的关系奠定了基础。

Cloning and expression of the metalloproteinase domain of human von Willebrand factor-cleaving protease

AIM: To clone and express the metalloproteinase domain of human von Willebrand factor-cleaving protease (vWF-cp). METHODS: The metalloproteinase domain of human vWF-cp, amplified from the plasmid containing the vWF-cp cDNA gene by using polymerase chain reaction, was cloned into pUC18, and its accuracy was verified by sequencing. Then the domain was inserted into the multiclone site of pET28a(+) and included a 6×His Tag at its amino terminal. After induced by IPTG, the recombinant protein was purified by using a Ni-NTA column and confirmed by Western blot. RESULTS: Comparison of the nucleotide sequence of our cloned domain with the GenBank sequence revealed no difference. High-level expression of the recombinant protein was yielded after 5-hour induction, which amounted to 28% of total bacteria protein in inclusion body. Western blot demonstrated that it possessed high specificity. CONCLUSION: The metalloproteinase domain of vWF-cp was high efficiently expressed in Escherichia coli. This might contribute to the further study of the relationship between its structure and function.