Production of human tissue factor using the Pichia pastoris expression system.
Tissue factor plays an important role in the initiation of the blood coagulation cascade resulting in the formation of a fibrin clot. The extracellular domain of human tissue factor has been expressed in the protease-deficient strain of the methylotrophic yeast Pichia pastoris, SMD1168. Tissue factor was expressed with a human influenza hemagglutinin tag fused at the C-terminus under control of the regulatory sequences from the Pichia AOX1 gene. Expressed protein was secreted in a soluble form at levels of up to 10 mg L-1 and correct processing of the PHO1 signal sequence was confirmed by N-terminal amino acid sequence analysis. Tissue factor was produced in Pichia as three discrete forms which appeared as three bands in the range 37-45 kDa by SDS-PAGE. These were all recognized by an anti-tissue factor monoclonal antibody. Deglycosylation 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 host for producing biologically active recombinant tissue factor requiring little purification.
Austin
A J
AJ
Department of Chemical and Process Engineering, University of Newcastle upon Tyne, United Kingdom.
Jones
C E
CE
Heeke
G V
GV
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017931:DNA Primers; D006031:Glycosylation; D006801:Humans; D007526:Isoelectric Point; D010843:Pichia / Q000235:genetics*; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D013925:Thromboplastin / Q000235:genetics* / Q000302:isolation & purification
D017931:DNA Primers; D011994:Recombinant Proteins; D013925:Thromboplastin