人USP14蛋白在大肠杆菌中表达纯化的初步研究

A preliminary study: cloning,expression and purification of human deubiquitinating enzyme USP14

采用PCR技术从人胎脑cDNA文库中克隆了usp14(ubiquitin-specific peptidase 14)基因编码区全长序列。序列分析表明人usp14基因含有peptidase蛋白酶C19A保守区,将usp14基因与pET-28b(+)载体相连,构建表达载体pET28b/usp14;把该表达载体转入大肠杆菌Rosetta(DE3)pLyS,以Isopropyl-D-thiogalactopyranoside(IPTG)诱导25°C 6小时,USP14蛋白获得高效表达。对表达产物进行Western blotting检测,并用Ni-NTA亲和层析技术获得了纯化的人USP14蛋白,表达的目的蛋白大小约为56kDa。

The usp14 gene was cloned from cDNA library of human fetus brain. The total coding region is 1485bp, encoding a peptide with 494 amino acid residues, with molecular weight of 56kD and pI of 5.20. The human usp14 gene was cloned to Nhe I and Xho I sites of pET-28b（ ＋ ） to construct an expression plasmid pET28b-usp14. The expression plasmid was transformed to E. coli Rosetta （ DE3 ） pLyS, and induced by Isopropyl-D-thiogalactopyranoside （IPTG）. SDS-PAGE analysis confirmed that the human USP14 protein was highly expressed after 6h induction at 25℃. The product of expression was identified by Western blotting and the USP14 protein was purified to homogeneity. Eukaryotic expression of usp14 gene is in progression.