RECOMBINANT LEISHMANIA SURFACE GLYCOPROTEIN GP63 IS SECRETED IN THE BACULOVIRUS EXPRESSION SYSTEM AS A LATENT METALLOPROTEINASE

A gene encoding the Leishmania surface metalloproteinase, GP63, was mo dified using the polymerase chain reaction to obtain effective secreti on of recombinant GP63 (reGP63) in the baculovirus insect cell express ion system. The coding region for the N-terminal signal peptide (SP) o f GP63 was modified to resemble the SP for the GP67 envelope protein f rom the budded virus form of Autographa californica nuclear polyhedros is virus. To prevent processing at the C-terminus with a glycosyl phos phatidylinositol anchor and the subsequent membrane anchoring of reGP6 3 in insect cells, the coding region for a putative SP at the C-termin us of GP63 was deleted. The reGP63 protein was glycosylated and secret ed as a latent metalloproteinase in the baculovirus expression system. The reGP63 protein was purified from serum-free medium using concanav alin A lectin affinity chromatography, with a yield of 1 mg/l. The pur ified Leishmania reGP63 was secreted as a latent proteinase. Treatment of reGP63 with HgCl2 resulted in activation of full proteinase activi ty and a concomitant decrease in M(r). The mechanism of the activation of Leishmania reGP63 is consistent with that of other members of the family of matrix-degrading metalloproteinases.