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Published on: March 5, 2013
[Cloning, expression and biological characterization of hTFPI-2 gene]
De-Sheng Kong1, Hong-Shen Guo, Xu Cai
1Key Laboratory of Molecular Medicine, Ministry of Education, Shanghai Medical College, Fudan University, Shanghai 20032, China.
Objective:
To clone the human tissue factor pathway inhibitor-2 (hTFPI-2) gene and express it by using prokaryotic expression system.
Methods:
The hTFPI-2 coding region was obtained by RT-PCR from human placenta total RNA. The coding fragment was then inserted into prokaryotic expression vector pET19b and expressed in E. coli BL21 by IPTG induction. The produced inclusion bodies were dissolved by denaturalizing chemicals, purified by ion exchange chromatograph, and refolded in air to form proper disulfide bonds. Chromogenic and gelatin zymography methods were used to evaluate the inhibiting effects of hTFPI-2 on trypsin, plasmin and MMPs individually. The inhibitory activity of hTFPI-2 on fabrisarcoma was investigated by matrigel.
Results:
The coding fragment of hTFPI-2 was cloned successfully and the protein was expressed as inclusion bodies which account for 20% - 30% of total host protein. The refolded hTFPI-2 could inhibit the invasive ability of fibrisarcoma HT-1080 as well as activity of plasmin, trypsin and MMPs.
Conclusions:
The activated hTFPI-2 is obtained by using prokaryotic expressed system effectively.
