MOLECULAR-CLONING AND EXPRESSION OF BOTHROJARACIN, A POTENT THROMBIN INHIBITOR FROM SNAKE-VENOM

Bothrojaracin is a potent and selective thrombin inhibitor that has be en isolated from the venom of Bothrops jararaca. It does not interact with the catalytic site of the enzyme but binds to both anion-binding exosites 1 and 2 resulting in a potent inhibition of thrombin activity towards fibrinogen and platelets [Zingali, R. B., Jandrot-Perrus, M., Guillin, M. C. & Bon, C. (1993) Biochemistyr 32, 10794-10802]. Bothro jaracin is a 27-kDa protein composed of two disulfide-linked polypepti de chains, A and B, of 15 kDa and 13 kDa, respectively. The sequences of A and B chains determined by molecular cloning exhibit a high degre e of identity with other snake venom lectin-like proteins. In contrast to other ligands that interact with thrombin exosite 1, the amino aci d sequence of bothrojaracin does not contain an acidic sequence simila r to the C-terminal tail of hirudin. Expression of functional bothroja racin was achieved in COS cells upon transfection with two pcDNA3 vect ors containing the complete cDNAs. Recombinant bothrojaracin, which wa s secreted into the medium, was able to bind to and inhibit thrombin. When expressed alone, the B chain formed inactive dimers that were sec reted into the culture medium. In contrast, no bothrojaracin-related p rotein was detected in conditioned media from cells transfected with A chain.