PRODUCTION OF HUMAN TISSUE FACTOR USING THE PICHIA-PASTORIS EXPRESSION SYSTEM

Tissue factor plays an important role in the initiation of the blood c oagulation cascade resulting in the formation of a fibrin clot. The ex tracellular domain of human tissue factor has been expressed in the pr otease-deficient strain of the methylotrophic yeast Pichia pastoris, S MD1168. Tissue factor was expressed with a human influenza hemagglutin in tag fused at the C-terminus under control of the regulatory sequenc es from the Pichia AOX1 gene. Expressed protein was secreted in a solu ble form at levels of up to 10 mg L-1 and correct processing of the PH O1 signal sequence was confirmed by N-terminal amino acid sequence ana lysis. Tissue factor was produced in Pichia as three discrete forms wh ich appeared as three bands in the range 37-45 kDa by SDS-PAGE. These were all recognized by an anti-tissue factor monoclonal antibody. Degl ycosylation studies using Endo H showed that the three forms were the result of differences in glycosylation of the protein. The low levels of secreted proteins produced by P. pastoris make this an efficient ho st for producing biologically active recombinant tissue factor requiri ng little purification. (C) 1998 Academic Press.