Prokaryotic expression, purification and activity analysis of recombinant human serine protease inhibitor Hespintor Kazal Domain.
- Author:
Jie FENG
;
Yongzhi LUN
;
Yue LI
;
Huijuan WU
;
Baoming LI
;
Ling WEI
;
Xiaoli ZHANG
;
Xuelei WANG
;
Qing CHI
- Publication Type:Journal Article
- MeSH:
Escherichia coli;
genetics;
metabolism;
Genetic Vectors;
genetics;
Hep G2 Cells;
Humans;
Proteinase Inhibitory Proteins, Secretory;
Recombinant Proteins;
biosynthesis;
genetics;
Serine Peptidase Inhibitors, Kazal Type;
Serine Proteinase Inhibitors;
biosynthesis;
classification;
genetics
- From:
Chinese Journal of Biotechnology
2013;29(11):1607-1616
- CountryChina
- Language:Chinese
-
Abstract:
Hespintor is an unknown function protein that was got from hepatoblastoma cell lines HepG2 by suppression subtractive hybridization technique (SSH), sequence analysis showed that the protein is a new member of secretory type of Kazal type serine protease inhibitor (Serpin) family, and has high homology with esophageal cancer related gene 2 (ECRG2). The coding sequence of Hespintor's Kazal domain was subcloned into prokaryotic expression vector pET-40b(+), then transformed into Rosetta (DE3). A recombinant protein about 42 kDa in the form of inclusion body was optimization expressed by inducing with 0.25 mmol/L IPTG, 30 degrees C for 5 h. and its specificity was confirmed via Western blotting. The recombinant protein was purified by metal chelate affinity chromatography (MCAC) and anion-exchange chromatography. The preliminary experimental result showed that the recombinant protein can inhibit trysin hydrolysis activity specifically. The result clearly demonstrated that Hespintor, as a novel member of Serpin, would be valuable in developing anti-tumor agents.
