[Prokaryotic expression, purification and activity analysis of recombinant human serine protease inhibitor Hespintor Kazal Domain].
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.
Jie
J
Feng
Yongzhi
Y
Lun
Yue
Y
Li
Huijuan
H
Wu
Baoming
B
Li
Ling
L
Wei
Xiaoli
X
Zhang
Xuelei
X
Wang
Qing
Q
Chi
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D056945:Hep G2 Cells; D006801:Humans; D053491:Proteinase Inhibitory Proteins, Secretory; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D000074162:Serine Peptidase Inhibitors, Kazal Type; D015842:Serine Proteinase Inhibitors / Q000096:biosynthesis* / Q000145:classification / Q000235:genetics
D053491:Proteinase Inhibitory Proteins, Secretory; D011994:Recombinant Proteins; C473799:SPINK7 protein, human; D000074162:Serine Peptidase Inhibitors, Kazal Type; D015842:Serine Proteinase Inhibitors