Expression, purification and evaluation of N-terminal domain of AcAP5 with Factor Xa inhibitory activity  

AcAP5蛋白N端片段的表达、纯化与活性评价(英文)

作　　者：刘爱华[1] 朱元军[1] 刘晓岩[1] 王银叶[1] 

机构地区：[1]北京大学医学部药学院分子与细胞药理学系,北京100191

出　　处：《Journal of Chinese Pharmaceutical Sciences》2013年第3期268-271,共4页中国药学（英文版）

基　　金：National Technology Graveness Special Purpose Fund (Grant No. 2009zx09301-010)

摘　　要：Ancylostoma anticoagulant peptide 5 （AcAP5） is a strong inhibitor of human coagulation factor Xa （FXa）. The N-terminal residues （N40） of AcAP5 contains a domain that could combine with FXa. In order to determine whether N40 protein has FXa inhibitory effect, we cloned, expressed and purified the protein for activity evaluation. The DNA fragment coding N40 was amplified by PCR, cloned into pET-30a to construct recombinant plasmid pET30a-N40, and subsequently transformed into E. coli, BL21 （DE3）. Expression of N40 was induced by isopropyl ~3-D-l-thiogalactopyranoside （IPTG）, and the interest protein was identified by SDS-PAGE and purified using one-step nickel （Ni） affinity chromatography. Under the optimal expres- sion condition （0.05 mM IPTG for 6 h at 37 ℃）, the purity of N40 reached 90%. We also evaluated the inhibition activity of N40 protein on FXa, finding the ICso was 4.58× 10 5 mol/L, This study suggests the N40 of AcAP5 could combine with FXa to inhibit FXa activity.犬钩虫抗凝肽5(Ancylostoma anticoagulant peptide 5,AcAP5)N端40个氨基酸片段(N40)含有与FXa结合的结构域。为研究N40的FXa抑制作用,我们克隆、表达和纯化了N40,并检测其生物活性。用PCR扩增N40基因;将PCR产物克隆至原核表达载体pET-30a中,构建质粒pET30a-N40;将其转化入大肠杆菌E.coli.BL21(DE3)中,IPTG诱导目的蛋白表达,SDS-PAGE电泳鉴定,经过镍柱亲和层析纯化,用BCA法进行蛋白定量,测定该蛋白抑制FXa的活性。限制性酶切鉴定和基因测序结果显示重组质粒构建成功;SDS-PAGE结果显示目的蛋白在E.coli.BL21(DE3)中为可溶性表达,亲和层析纯化后获得了纯度约90%的蛋白,经活性鉴定该蛋白确有抑制FXa的活性。

关 键 词：AcAP5 FXa binding domain FXa inhibition EXPRESSION PURIFICATION 

分 类 号：Q51[生物学—生物化学]