1.Prokaryotic expression, purification and activity analysis of recombinant human serine protease inhibitor Hespintor Kazal Domain.
Jie FENG ; Yongzhi LUN ; Yue LI ; Huijuan WU ; Baoming LI ; Ling WEI ; Xiaoli ZHANG ; Xuelei WANG ; Qing CHI
Chinese Journal of Biotechnology 2013;29(11):1607-1616
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.
Escherichia coli
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genetics
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metabolism
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Genetic Vectors
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genetics
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Hep G2 Cells
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Humans
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Proteinase Inhibitory Proteins, Secretory
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Recombinant Proteins
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biosynthesis
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genetics
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Serine Peptidase Inhibitors, Kazal Type
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Serine Proteinase Inhibitors
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biosynthesis
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classification
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genetics