罗氏沼虾雄性生殖系统Kazal型蛋白酶抑制剂及精子明胶水解酶的原核表达及纯化
Prokaryotic Expression，Purification of Recombinant Male Reproduction-Related Peptidase Inhibitor Kazal-Type (MRPINK) and Sperm Gelatinase (MSG), Macrobrachium rosenbergii
在以前的研究中，我们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（MRPINK）和一种精子明胶水解酶（MSG）的cDNA全序列。本文将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IPTG诱导后，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和15.7 kDa, 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的分离纯化，得到了高纯度的表达蛋白。研究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
In our 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×His 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 the molecular weight of rMRPINK and rMSG were 14.7 and 15.7 kDa with the expected size, respectively, and both of them 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
俞炎琴、杨卫军、钱叶青、李晔
生物工程学分子生物学生物化学
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
Macrobrachium rosenbergiimale reproduction-related peptidase inhibitorKazal- type peptidase inhibitorsperm gelatinaseprokaryotie expressionpurification
俞炎琴,杨卫军,钱叶青,李晔.罗氏沼虾雄性生殖系统Kazal型蛋白酶抑制剂及精子明胶水解酶的原核表达及纯化[EB/OL].(2009-01-09)[2025-08-02].http://www.paper.edu.cn/releasepaper/content/200901-418.点此复制
评论