Cloning, expression and biological characterization of hTFPI-2 gene.
- Author:
De-Sheng KONG
1
;
Hong-Shen GUO
;
Xu CAI
;
Wang LIANG
;
Zhuo-Chun PENG
;
Hou-Yan SONG
;
Duan MA
Author Information
- Publication Type:Journal Article
- MeSH: Cloning, Molecular; Escherichia coli; metabolism; Gene Expression; Glycoproteins; biosynthesis; genetics; Humans; Placenta; cytology; RNA; isolation & purification; Reverse Transcriptase Polymerase Chain Reaction
- From: Chinese Journal of Hematology 2006;27(9):606-610
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo clone the human tissue factor pathway inhibitor-2 (hTFPI-2) gene and express it by using prokaryotic expression system.
METHODSThe 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.
RESULTSThe 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.
CONCLUSIONSThe activated hTFPI-2 is obtained by using prokaryotic expressed system effectively.
