Ll. Button et al., RECOMBINANT LEISHMANIA SURFACE GLYCOPROTEIN GP63 IS SECRETED IN THE BACULOVIRUS EXPRESSION SYSTEM AS A LATENT METALLOPROTEINASE, Gene, 134(1), 1993, pp. 75-81
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.