| Autor: |
Austin, Angela J., Jones, Carol E., Van Heeke, Gino |
| Zdroj: |
Protein Expression and Purification; June 1998, Vol. 13 Issue: 1 p136-142, 7p |
| Abstrakt: |
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 yeastPichia pastoris,SMD1168. Tissue factor was expressed with a human influenza hemagglutinin tag fused at the C-terminus under control of the regulatory sequences from thePichia AOX1gene. Expressed protein was secreted in a soluble form at levels of up to 10 mg L−1and correct processing of thePHO1signal sequence was confirmed by N-terminal amino acid sequence analysis. Tissue factor was produced inPichiaas 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 byP. pastorismake this an efficient host for producing biologically active recombinant tissue factor requiring little purification. |
| Databáze: |
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