Expression and purification of the recombinant human tissue factor in Pichia pastoris

Objective To obtain recombinant human extracellular tissue factor(r-TF) which can be used to set up PT kit. Methods Expression plasmid,pPIC9K-TF,was constructed by inserting the sequence encoding human extracellular tissue factor into yeast expression vector pPIC9K and transformed into Pichia pastoris GS115 with electroporation.Having been selected by G418,transformants containing TF cDNA were induced by methanol for the expression of rTF,purificated and anlaysis activity. Results SDS-PAGE showed that the molecular weight of the expression product was about 37 000-45 000.Western-blotting indicated that it was human extracellular tissue factor.After phorspholipids treatment,purified rTF was able to initiate blood coagulation. Conclusions rTF gene is expressed in Pichia pastoris and the active products are secreted into the medium with concentration up to 182 mg/L.The recombinant protein is purified with a simple process and high rate of protein recovery,the purified protein can be used in PT test.