Cloning, Expression And Functional Study Of Tissue Factor Pathway Inhibitor-2 And Its First Kunitz Domain

Tissue factor pathway inhibitor-2 (TFPI-2), also known as placenta protein-5 (PP5) or matrix associated serine protease inhibitor (MSPI), is a protease inhibitor with Kunitz domains and belongs to serine protease inhibitor superfamily. Because the structure is homologous to TFPI (tissue factor pathway inhibitor), an important regulator of extrinsic pathway coagulation, TFPI-2 has got its name. 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, cathepsin G, and plasma kallikrein but not FVIIa/TF (tissue factor), FXa, t-PA (tissue-type plasminogen activator) and uPA (urokinase-type plasminogen activator).The three kunitz domains of TFPI have different functions. For example, KD1 (kunitz domain1) can bind and inhibit FVIIa/TF activity; KD2 can bind and inhibit FXa activity; KD3 interacts with heparin, CD14, LRP (low density lipoprotein receptor-related protein), and gp330. Furthermore, KD3 has several other functions such as anti-inflammation, coagulation assistant and TFPI metabolism. This hints that each domain of TFPI-2 may have individual functions and lots of experiments must be performed to validate the hypothesis.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 these, a novel drug which maybe used to prevent tumor's invasion and atherosclerosis could be developed from hTFPI-2.Up to now, the physiological roles of TFPI-2 have not been known, which significantly limits its applications. As a protease inhibitor in extracellular matrix (ECM), TFPI-2 plays an important role in regulating ECM remodeling. Consequently, it is thought that TFPI-2 may have close relations with cells migration, embryo development, wound healing, tumor invasion, atherosclerosis and so on. Geneknocking out in model animals using recombination and RNAi has been a powerful tool to investigate a gene's function. In light of the progression of TFPI-2 research, it is necessary to study TFPI-2 physiological functions by using model animals to give more theory to the protein's research and applications.We obtained the cDNA of hTFPI-2 using RT-PCR from human placenta total RNA and cloned it into prokaryotic expression vector pET19b. E.coli BL21 was used as host to express hTFPI-2 and the product which formed inclusion bodies reached 20%-30% of total host protein. Followed purification and refolding, the purity of the target protein reached to 90% and the yield was 20-30 mg/L. By biological characterizations research, we found the refolded hTFPI-2 could inhibit invasive ability of fibrisarcoma HT-1080 cells as well as activity of plasmin, trypsin, MMP-2 and MMP-9.This article also investigated the production procedure of hTFPI-2/KDl by using yeast expression system and the characterization and structure of the domain. At the beginning of the research, we cloned the wild-type coding region of hTFPI-2/KDl and transfected it into pichia pastoris, but the expression was not satisfactory. After analyzing the features of the codon, we found that there are so many yeast rare codons in the cDNA of TFPI-2/KD1. Then, we thought that the codon preference should be one of restrictions to the expression of hTFPI-2/KD1 in yeast. So, we synthesized hTFPI-2/KD1's gene according to the codon preference of pichia pastoris and cloned it into the vector pPIC9K. Then, the recombinant vector transformed into pichia pastoris was performed by electroporation and multicopy transformant was screened out successfully. Fermentation was carried out in a 30 L working volume stirred-tank reactor and the production peak was reached 40hrs after induction by methanol. The cultured medium was purified by ultrafiltration, gel filtration and ion exchange chromatography successively. Following this process, the purity of target protein reached 96% and the yield was 20 mg/L. By characterization research, we found that hTFPI-2/KDl could inhibit plasmin, trypsin, MMP-2 and MMP-9 activity. In the study, the inhibition of hTFPI-2/KD1 to the Matrigel invasion by HT-1080 was also described.We cloned cDNA of zebrafish TFPI-2 (zTFPI-2) successfully by using zebrafish gene bank, bioinformation, RT-PCR and RACE (rapid amplication of cDNA ends) method. After analyzing the amino acid sequence of zTFPI-2, we found zTFPI-2 also consisted of three tandem Kunitz domains which show 59% , 65% and 51%similarities with counterpart of hTFPI-2 respectively. Whole mount in situ hybridization showed that zTFPI-2 was mainly expressed in head and pectoral fins of early zebrafish embryo; Real time PCR showed that zTFPI-2 expression level was sequential decreased in adult fish brain, heart, liver, eyes and ovary. The results hinted that zTFPI-2 might influence the functions or development of these organs. To elucidate its function, Morpholino oligos was synthesized according to the zTFPI-2 gene. When the zTFPI-2 expression was downregulated by the Morpholino, the Morphant showed small head, brain white matter sparseness, big yolk-sac, pericardial sac edema and development retardation of pectoral fins. By microarray analysis, we found that there are about six hundreds of genes upregulated or downregulated by 2 or more folds. The changed genes included structure molecules, enzymes, second signal transducers, transcript factors and so on. Among these, Notch signal pathway members such as Notch la, mib and her4, which play important roles in central nervous system development were also affected obviously and the results were verified by real-time PCR. These finding will be a good commence for study the molecular mechanisms of zTFPI-2 influence on central nervous system development.