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罗氏沼虾Kazal型蛋白酶抑制剂及精子水解酶的原核表达和纯化

发表时间:2009-10-15  浏览量:1616  下载量:576
全部作者: 钱叶青,李晔,俞炎琴,杨卫军
作者单位: 浙江大学生命科学学院细胞生物与遗传研究所
摘 要: 以前的研究中,克隆获得了一种罗氏沼虾雄性生殖系统Kazal型蛋白酶抑制剂(male reproduction-related peptidase inhibitor Kazal-type, MRPINK)和一种精子明胶水解酶(Macrobrachium rosenbergii sperm gelatinase, MSG)的cDNA全序列。实验将MRPINK和MSG基因克隆到带有6×His标签的原核表达载体pET-28a(+)中,经酶切和测序鉴定得到正确的重组质粒pET-28a(+)-MRPINK和pET-28a(+)-MSG,转化E� coli BL21(DE3)后获得表达菌株。该表达菌株经异丙基硫代半乳糖苷(IPTG)诱导,高效表达了重组蛋白。软件预测重组蛋白rMRPINK和rMSG分子量分别为14�7,15�7 ku, SDS-PAGE结果显示与预测值一致。可溶性分析表明:rMRPINK和rMSG均表达在包涵体中,利用Ni-NTA纯化系统,这两种重组蛋白在变性条件下被有效地分离纯化,得到了高纯度的表达蛋白。为进一步研究抑制剂和蛋白酶的相互作用特点及其生理功能提供了基础。
关 键 词: 生殖生物学;罗氏沼虾;雄性生殖系统;Kazal型蛋白酶抑制剂;精子明胶水解酶;原核表达;纯化
Title: Prokaryotic expression, purification of recombinant Kazal-type peptidase inhibitor and sperm gelatinase, Macrobrachium rosenbergii
Author: QIAN Yeqing, LI Ye, YU Yanqin, YANG Weijun
Organization: Institute of Cell Biology and Genetics, College of Life Sciences, Zhejiang University
Abstract: In previous reports, full-length cDNAs of a male reproduction-related peptidase inhibitor Kazal-type (MRPINK) and a sperm gelatinase (MSG) have been identified from the prawn, Macrobrachium rosenbergii. The expression vector pET-28a(+)-MRPINK and pET-28a(+)-MSG were constructed by inserting MRPINK and MSG cDNA into 6譎is tag pET-28a(+), respectively. Through digestion with restriction enzymes and sequence analysis, the right expression strain were selected after transformation of recombined plasmid into E. coli BL21 (DE3) and the fusion proteins with His-tag were efficiently expressed after IPTG induction. SDS-PAGE analysis revealed that the molecular weight of rMRPINK and rMSG were 14.7 and 15.7 ku with the expected size, respectively, and both of them were expressed in inclusion bodies. Using Ni-NTA purification system, these recombinant proteins were efficiently purified under denature conditions and purified protein can be used in the further study of inhibitor and enzyme interaction mode and physiological functions.
Key words: biology of reproduction; Macrobrachium rosenbergii; male reproduction-related peptidase inhibitor; Kazal-type peptidase inhibitor; sperm gelatinase; prokaryotic expression; purification
发表期数: 2009年10月第19期
引用格式: 钱叶青,李晔,俞炎琴,等. 罗氏沼虾Kazal型蛋白酶抑制剂及精子水解酶的原核表达和纯化[J]. 中国科技论文在线精品论文,2009,2(19):2022-2027.
 
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