Study On Large-scale Fermentation And Purification Of Recombinant Human Tissue Factor Pathway Inhibitor 2

Tissue factor pathway inhibitor-2 is a recently discovered serine protease inhibitor, the same as placental protein-5 and matrix- associated serine protease inhibitor. In vitro essay, TFPI-2 can inhibit fibrinolysin, trypsinase matrix, metalloproteinase-1, MMP-3 and so on. It plays an important role in cell matrix remodeling, Angiogenesis, regulating invasion and metastasis of tumor cell, also involved in thrombokinesis and apoptosis.It is great interested in the research of the relationship between TFPI-2 and tumor cell's invasion and metastasis. Since the tumor cell can produce and secrete many kinds of serine protease inhibitor such as fibrinolysin, trypsinase matrix, collagenase, cathepsin which could degrade most matrix substance, then destroy extracellular matrix in order to facilitate tumor cell invasion and metastasis. TFPI-2 could obviously inhibit the invasion and metastasis of tumor cell including chorionic carcinoma cell, melanoma cell, prostatic carcinoma cell, pulmonary carcinoma cell and neuroglioma cell. The investigation of the relationship between TFPI-2 and tumor cell's invasion and metastasis provides new therapy of cancer cell. So we need rhTFPI-2 for further research, if apply to cancer therapy, great amount will be needed. 1. Studies on large-scale fermentation and purification process of rhTFPI-2. Studies on large-scale fermentation process of rhTFPI-2: Pichia pastoris is a monocellular eukaryon which has many advantages as an expression host, and it's suitable for large-scale expression of the extraneous proteins. The large-scale fermentation process in 80L fermentor of rhTFPI-2 was explored. Studies were focused on pH value, culture medium, resolved oxygen, methanol feed speed, initial biomass etc. The best conditions for fermentation were found as follows: FM21 medium (including 0.5% peptone), yeast biomass 200g?L-1, pH 6.2, DO between 20～30% and the supply speed of methanol 6～7ml·h-1·L-1 initial fermentation volume. The concentration of rhTFPI-2 in the broth could be reached 970mg?L-1.2. A new method to purify rhTFPI-2 at large-scale: The fermentation supernatant was purified with SP Sepharose XL cation exchange chromatogr aphy, collecting the eluate at 0.6mol·L-1 NaCl. Then the eluate was sampled on hydrophobic chromatography column and distilled to condense rhTFPI-2. The purity could be more than 95% and the yield coefficient was higher than 58%..Above all, this assay first time explored the fermentation condition of rhTFPI-2 Pichia pastoris at large-scale in 80L fermentor. Lay the foundation for Industrial production in the next step and the clinical application to further explore rhTFPI-2.