Expression of functional coagulation factor XIII in Escherichia coli.
Coagulation factor XIII is a zymogen that can be activated by thrombin cleavage to a transglutaminase that catalyses the formation of covalent crosslinks between fibrin chains in the final stages of the blood clotting cascade. Although circulating factor-XIII is composed of A and B subunits the catalytic activity is a property of the A subunits. In this study we have constructed a plasmid (pKKF13A) that contains a cDNA encoding the A subunit positioned downstream of a tac promoter. Escherichia coli containing this plasmid produce A subunit protein when grown in the presence of IPTG. The cloned A subunit has been partially purified and characterized. Comparison with A subunits purified from plasma showed that the cloned A subunits were of the same size, assembled as dimers, and had the same native electrophoretic mobility. The cloned A subunits expressed transglutaminase activity with putrescine, dansylcadaverine and casein as substrates, and were able to crosslink fibrin in clots formed from A subunit deficient plasma. These studies have demonstrated that functional recombinant factor XIII A subunit can be produced in E. coli and suggest that recombinant factor XIII can potentially provide a safe and inexhaustible supply for therapeutic use.
Board
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PG
Human Genetics Group, John Curtin School of Medical Research, Australian National University, Canberra.
Pierce
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K
Coggan
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M
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D004926:Escherichia coli / Q000378:metabolism*; D005176:Factor XIII / Q000096:biosynthesis* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D011503:Transglutaminases
D011993:Recombinant Fusion Proteins; D005176:Factor XIII; D011503:Transglutaminases