Related Experiment Videos
Production of human tissue factor using the Pichia pastoris expression system
A J Austin1, C E Jones, G V Heeke
1Department of Chemical and Process Engineering, University of Newcastle upon Tyne, United Kingdom.
Protein Expression and Purification
|June 19, 1998
Summary
Researchers successfully expressed human tissue factor in Pichia pastoris yeast. This method efficiently produces biologically active recombinant tissue factor for potential therapeutic applications.
Area of Science:
- Biochemistry
- Molecular Biology
- Biotechnology
Background:
- Tissue factor is crucial for initiating the blood coagulation cascade and fibrin clot formation.
- Recombinant protein expression in yeast offers a scalable production system.
Purpose of the Study:
- To express the extracellular domain of human tissue factor in Pichia pastoris.
- To assess the yield, solubility, and biological activity of the recombinant protein.
Main Methods:
- Human tissue factor gene cloned into Pichia pastoris under AOX1 promoter control.
- Protein expression, secretion, N-terminal sequencing, SDS-PAGE, and deglycosylation (Endo H) were performed.
- Characterization using anti-tissue factor monoclonal antibody.
Main Results:
- Soluble recombinant human tissue factor secreted at levels up to 10 mg/L.
- Correct signal sequence processing confirmed by N-terminal sequencing.
- Three distinct protein forms (37-45 kDa) observed, differing in glycosylation.
- All forms recognized by anti-tissue factor antibody.
Conclusions:
- Pichia pastoris is an efficient host for producing biologically active recombinant human tissue factor.
- The yeast system yields high levels of secreted protein, simplifying purification.
- The produced tissue factor has potential for therapeutic and research applications.