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Expression of functional coagulation factor XIII in Escherichia coli
P G Board1, K Pierce, M Coggan
1Human Genetics Group, John Curtin School of Medical Research, Australian National University, Canberra.
Thrombosis and Haemostasis
|April 12, 1990
Summary
Researchers produced functional recombinant coagulation factor XIII A subunit in E. coli. This breakthrough offers a potential safe and abundant source for therapeutic applications in blood clotting disorders.
Area of Science:
- Biochemistry
- Molecular Biology
- Hematology
Background:
- Coagulation factor XIII (FXIII) is essential for blood clot stabilization.
- FXIII's catalytic activity resides in its A subunits, which function as a transglutaminase.
- Current FXIII sources may have limitations in safety and supply.
Purpose of the Study:
- To develop a method for producing functional recombinant FXIII A subunit.
- To characterize the recombinant A subunit produced in Escherichia coli (E. coli).
- To assess the potential of recombinant FXIII for therapeutic use.
Main Methods:
- Constructed a plasmid (pKKF13A) with cDNA encoding the FXIII A subunit under a tac promoter.
- Induced expression of the A subunit in E. coli using IPTG.
- Partially purified and characterized the recombinant A subunit, comparing it to plasma-derived A subunits.
- Assessed transglutaminase activity using various substrates and fibrin crosslinking capability.
Main Results:
- E. coli successfully produced the FXIII A subunit protein when induced.
- The cloned A subunits were similar in size, assembled as dimers, and exhibited identical native electrophoretic mobility to plasma-derived A subunits.
- Recombinant A subunits demonstrated transglutaminase activity and effectively crosslinked fibrin.
Conclusions:
- Functional recombinant factor XIII A subunit can be produced in E. coli.
- Recombinant FXIII holds promise as a safe and potentially inexhaustible therapeutic agent for coagulation disorders.