野生型和功能增强型ADAMTS13蛋白酶分子的表达及功能鉴定

Expression and functional identification of WT-/GOF-ADAMTS13 proteins

背景:血管性血友病因子水解蛋白酶13(a disintegrin and metalloproteinase with a thrombospondin type 1motif,member 13,ADAMTS13)可结合并酶切血管性血友病因子,在防止血小板过多聚集和血栓的形成中起到关键作用。重组ADAMTS13通常选择在真核细胞中表达分泌,然而对于其表达纯化目前仍缺乏一个明确高效的途径。目的:寻找合适的宿主细胞,得到稳定表达野生型ADAMTS13和功能增强型ADAMTS13的细胞株,建立高纯度ADAMTS13的纯化方法,进而研究其生物学功能。方法:以野生型ADAMTS13重组质粒为模板,利用位点突变试剂盒得到功能增强型ADAMTS13重组质粒。比较CHO-S细胞、CHO-K1细胞、293T细胞对ADAMTS13的表达效果,挑选出合适的宿主细胞。通过Ni柱亲和层析和分子筛纯化蛋白;采用7.5%SDS-PAGE、Western-blot鉴定野生型ADAMTS13和功能增强型ADAMTS13的纯度及特异性;利用原子力显微镜技术检测野生型及功能增强型ADAMTS13与血管性血友病因子A2的相互作用,鉴定两种蛋白的活性。结果与结论:相对于CHO-S和CHO-K1,293T细胞更适合作为野生型ADAMTS13和功能增强型ADAMTS13的表达宿主;经Ni柱亲和层析和分子筛纯化后,所得到的野生型ADAMTS13和功能增强型ADAMTS13质量浓度分别为103.7,149.7 mg/L,且两种蛋白纯度均约90%;原子力显微镜检测结果显示,野生型ADAMTS13、功能增强型ADAMTS13与血管性血友病因子A2的黏附频率分别为11.37%,14.70%,表明功能增强型ADAMTS13与血管性血友病因子A2的结合亲和力更高,这与近期ADAMTS13构象研究一致。

BACKGROUND:ADAMTS13 cleaves Von Willebrand factor(VWF)to regulate its size,thereby preventing aberrant platelet aggregation and thrombus.Eukaryotic cells are usually selected to express recombinant ADAMTS13.However,it still lacks a clear and efficient way for its expression and purification.OBJECTIVE:To find a suitable host cell and get stable cell lines to express WT-ADAMTS13(wild-type)and GOF-ADAMTS13(gain of function),so as to establish the purification method for high-purity ADAMTS13 proteins,thus studying their biological functions.METHODS:GOF-ADAMTS13 recombinant plasmid was obtained by site mutation kit with the WT-ADAMTS13 recombinant plasmid as a template.The expression effects of CHO-S,CHO-K1,and 293T cells on ADAMTS13 were compared to select appropriate host cells.The proteins were purified by Ni affinity chromatography and gel filtration.The purity and specificity of WT-ADAMTS13 and GOF-ADAMTS13 were identified by 7.5%SDS-PAGE and western blot assay.The interaction between WT-/GOF-ADAMTS13 and VWF-A2 was detected by atomic force microscopy to validate the activity of the two proteins.RESULTS AND CONCLUSION:Compared with CHO-S and CHO-K1 cell lines,293T cell line was more suitable as expression host for WT-/GOF-ADAMTS13.After Ni column affinity chromatography and gel filtration purification,the concentration of purified WT-/GOF-ADAMTS13 was 103.7 and 149.7 mg/L,respectively with the purity of above 90%.Atomic force microscopy results showed that the adhesion frequencies of WT-/GOF-ADAMTS13 and VWF-A2 were 11.37%and 14.70%,respectively.These results suggest higher affinity of GOF-ADAMTS13 binding to VWF-A2 is consistent with recent ADAMTS13 conformational studies.