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 modified using the polymerase chain reaction to obtain effective secretion of recombinant GP63 (reGP63) in the baculovirus insect cell expression system. The coding region for the N-terminal signal peptide (SP) of GP63 was modified to resemble the SP for the GP67 envelope protein from the budded virus form of Autographa californica nuclear polyhedrosis virus. To prevent processing at the C-terminus with a glycosyl phosphatidylinositol anchor and the subsequent membrane anchoring of reGP63 in insect cells, the coding region for a putative SP at the C-terminus of GP63 was deleted. The reGP63 protein was glycosylated and secreted as a latent metalloproteinase in the baculovirus expression system. The reGP63 protein was purified from serum-free medium using concanavalin A lectin affinity chromatography, with a yield of 1 mg/l. The purified Leishmania reGP63 was secreted as a latent proteinase. Treatment of reGP63 with HgCl2 resulted in activation of full proteinase activity 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.
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Department of Medical Genetics, Faculty of Medicine, University of British Columbia, Vancouver, Canada.
Wilson
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Astell
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McMaster
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WR
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D016895:Culture Media, Serum-Free; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006031:Glycosylation; D018320:Leishmania major / Q000201:enzymology* / Q000235:genetics; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008666:Metalloendopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009036:Moths; D021382:Protein Sorting Signals / Q000235:genetics; D015800:Protozoan Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D016895:Culture Media, Serum-Free; D004274:DNA, Recombinant; D008562:Membrane Glycoproteins; D021382:Protein Sorting Signals; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C055146:glycoprotein gp63, Leishmania