Htfpi Recombinant Protein Expression Conditions Optimized

Tissue factor (TF)-initiated coagulation plays a significant role in the development ofmany diseases, including cancer, cardiovascular diseases and inflammation, whichmay lead to the death of some patients. Human tissue factor pathway inhibitor(hTFPI) is one of the major physiological inhibitors of the extrinsic coagulationpathway initiated by TF. Numerous evidences showed that rhTFPI is a potentialcandidate for the prevention and cure of many diseases, such as thrombosis, infectiveshock, vascular restenosis, endo toxemia and disseminated intravascular coagulation(DIC), etc.To date, rhTFPI has been expressed in several protein expression systems, and themain problem still remained was the low level of expression. The highest expressionwas achieved in some mammalian cell lines (about 2-5mg/L), while the cost was alsothe highest. Pichia Pastoris expression system was chosen to express rhTFPI in thisstudy. The results showed that Pichia pastoris expressed rhTFPI at the level of 1.6mg/L in shaking flask and at the level of 4.6 mg/L under the condition of high densityfermentation culture. The methods for the purification of rhTFPI from the culturemedium were optimized. The rhTFPI purified using the optimized protocol(ammonium sulfate fractionation plus anion exchange chromatography and plus gelfiltration chromatography) has a purity of more than 95ï¼…with the recovery rate being43.1ï¼…. The advantages of expressing rhTFPI in Pichia pastoris expression system overthat in mammalian expression systems are the significantly lower cost for cell cultureand protein purification. When compared with E.coli. protein expression system,Pichia pastoris has a higher level expression of hTFPI and the resulting TFPI is moreresembled to the native TFPI in term of the posttranslational modification.