AcAP5蛋白N端片段的表达、纯化与活性评价

doi:10.5246/jcps.2013.03.038

AcAP5蛋白N端片段的表达、纯化与活性评价

刘爱华, 朱元军, 刘晓岩, 王银叶^*

北京大学医学部药学院分子与细胞药理学系, 北京 100191

收稿日期:2012-11-05 修回日期:2013-02-26 出版日期:2013-05-08 发布日期:2013-05-08
通讯作者: 王银叶*

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

Aihua Liu, Yuanjun Zhu, Xiaoyan Liu, Yinye Wang^*

Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China

Received:2012-11-05 Revised:2013-02-26 Online:2013-05-08 Published:2013-05-08
Contact: Yinye Wang*

摘要/Abstract

摘要： 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: 犬钩虫抗凝肽5, FXa结合结构域, FXa抑制作用, 表达, 纯化

Abstract:

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 β-D-1-thiogalactopyranoside (IPTG), and the interest protein was identified by SDS-PAGE and purified using one-step nickel (Ni) affinity chromatography. Under the optimal expression condition (0.05 mM IPTG for 6 h at 37 °C), the purity of N40 reached 90%. We also evaluated the inhibition activity of N40 protein on FXa, finding the IC50 was 4.58×10^-5mol/L. This study suggests the N40 of AcAP5 could combine with FXa to inhibit FXa activity.

Key words: AcAP5, FXa binding domain, FXa inhibition, Expression, Purification

中图分类号:

R962.1

Supporting:

Foundation item: National Technology Graveness Special Purpose Fund (Grant No. 2009zx09301-010).
^*Corresponding author. Tel.: 86-10-82802652; Fax: 86-10-62015584

刘爱华, 朱元军, 刘晓岩, 王银叶^*. AcAP5蛋白N端片段的表达、纯化与活性评价[J]. , DOI: 10.5246/jcps.2013.03.038.

Aihua Liu, Yuanjun Zhu, Xiaoyan Liu, Yinye Wang^*. Expression, purification and evaluation of N-terminal domain of AcAP5 with Factor Xa inhibitory activity[J]. , DOI: 10.5246/jcps.2013.03.038.

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