拟南芥RRP41L蛋白的原核表达与纯化  

Prokaryotic expression and purification of RRP41L in Arabidopsis thaliana

作　　者：杨敏[1] 王翠玲[1] 侯小改[1] 

机构地区：[1]河南科技大学农学院,河南洛阳471003

出　　处：《河南农业大学学报》2014年第6期731-735,共5页Journal of Henan Agricultural University

基　　金：国家青年科学基金项目(31101153)

摘　　要：采用PCR技术克隆得到拟南芥类核糖体RNA加工蛋白41(Ribosomal RNA-processing protein 41-LIKE,RRP41L)基因,然后构建其原核表达载体,并对其进行诱导和纯化,以期获得较大量的重组蛋白.结果表明,试验获得了拟南芥RRP41L基因的全长编码序列(coding sequence,CDS)(771 bp),将其与原核表达载体PGEX4T-1连接构建了原核表达载体PGEX4T-RRP41L,将PGEX4T-RRP41L质粒导入大肠杆菌(E.coli)BL21(DE3)中,在37℃条件下,用1 mmol·L-1IPTG诱导8 h后体外纯化融合蛋白.SDS-PAGE电泳结果显示,在此诱导条件下,PGEX4T-RRP41L重组质粒可表达出较大量的重组蛋白,蛋白质相对分子质量大小为53.4 k D,除去PGEX4T-1自身表达相对分子质量大小为26.0 k D后,与RRP41L编码的蛋白质相对分子质量约为27.4 k D的大小一致.In order to construct prokaryotic expression vector and to obtain the recombinant gene ex- pression in the host bacteria, we cloned full-length coding sequence （CDS） of Ribosomal RNA-pro- eessing Protein 41-LIKE （RRP41 L） in Arabidopsis by the PCR method followed by restriction enzymes cutting and connection. The results of the experiments showed： the full-length of RRP41L CDS （771 bp） was cloned, fused with the vector of PGEX4T-1, and a recombinant of prokaryotic expression vector （PGEX4T-RRP41L） was constructed successfully. Then the expression plasmid PGEX4T- RRP41L was transformed to E. coli BL21 （DE3）. RRP41L protein expressed effectively after being in- duced for 8 hours with 1 mmol·L^-1 IPTG at 37℃. The SDS-PAGE results indicated that the fusion protein was expressed with molecular weight of 53.4 kD, with the PGEX4T-1 own induction produced 26.0 kD protein. The result was consistent with the 27.4 kD protein which encoded by RRP41L.

关 键 词：拟南芥 类核糖体RNA加工蛋白41 原核表达 蛋白纯化 

分 类 号：Q202[生物学—细胞生物学]