Human Tissue Factor Pathway Inhibitor-2 Domain Of 1, 3 And C-terminal Expression, Purification And Structure And Function

Tissue factor pathway inhibitor-2(TFPI-2) is a protease inhibitor with Kunitz domains and belongs to serine protease inhibitor superfamily.The protein consists of five parts:a short acidic amino-terminal region,three tandem Kunitz domains and a carboxyl-terminal tail highly enriched in basic amino acids.Experiments in vitro showed that human TFPI-2(hTFPI-2) could inhibit several proteases activity including trypsin, plasmin,chymotrypsin,plasma kallikrein,and cathepsin G.Several studies revealed that hTFPI-2 could inhibit invasive ability of many tumors such as prostate cancer,lung cancer,fibrosarcoma,melanoma and gliomas,et al.Furthermore,the rupture of atherosclerosis plaque can also be inhibited by hTFPI-2.Because of this,a novel drug which maybe used to prevent tumor's invasion and atherosclerosis could be developed from hTFPI-2.But up to now,the physiological roles of TFPI-2 is not clear yet,which significantly limits its applications.It is believed that the first kunitz-type domain of hTFPI-2(hTFPI-2/KD1) harbors most of hTFPI-2's inhibitory activities.That's why it is important to study the structure-function relationship.In this article,the conformation of Kunitz domain 1 of human tissue factor pathway inhibitor-2(hTFPI-2/KD1) has been studied by Fourier transform infrared spectroscopy,circular dichroism,and Raman spectroscopy.It was found that hTFPI-2/KD1 contained approximately 17%α-helices,24%β-strands,46% random coils,13%β-turns,and two kinds of disulfide bonds(ggg and tgt) at 25℃.The detailed conformational changes of the heated protein observed by FTIR,CD and Raman spectroscopy revealed that hTFPI-2/KD1 was thermally stable.However,KD1 could form an intermediate form at high temperature,and then return to its normal conformation when the temperature was lowered.Activity assays also showed that hTFPI-2/KD1 was able to keep its inhibitory activity on plasmin after being heated to 80℃for 5 min.In this article we also investigated the preperation procedure of hTFPI-2/KD3C by using yeast expression system and the characterization of the domain.After the hTFPI-2/KD3C's gene was synthesized according to the codon preference of pichia pastoris,and the glycosylation site was mutated by the laboratory,the synthesized gene was cloned into the vector pPIC9K.Then,the recombinant vector transformed into pichia pastoris was performed by electroporation and multicopy transformant was screened out successfully.Through fermentation,expression and purification,the purity of target protein reached 97%and the yield was 25 mg/L.The first ~1H NMR experiment of hTFPI-2/KD3C has been carried out on a Bruker DMX 500 spectrometer.According to the high sequence similarity between the KD3C of hTFPI-2 and hTFPI-1,we made an initial exploration on the structure-function of hTFPI-2/KD3C,using hTFPI-1/KD3C as a reference.We found that hTFPI-2/KD3C could bind to heparin and the NaCl can block their binding.Its binding sites with heparin have also been discussed.