Recombinant Leishmania surface glycoprotein GP63 is secreted in the baculovirus expression system as a latent metalloproteinase
| Autor: | Caroline R. Astell, Wilson G, L L Button, W R McMaster |
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| Rok vydání: | 1993 |
| Předmět: |
Signal peptide
Glycosylation Molecular Sequence Data DNA Recombinant Protozoan Proteins Moths Protein Sorting Signals Chromatography Affinity Culture Media Serum-Free law.invention law parasitic diseases Gene expression Genetics Animals Secretion Amino Acid Sequence Cloning Molecular Cells Cultured Leishmania major chemistry.chemical_classification Metalloproteinase Membrane Glycoproteins Base Sequence biology Metalloendopeptidases General Medicine biology.organism_classification Molecular biology Recombinant Proteins Enzyme Activation Autographa californica Biochemistry chemistry Concanavalin A Recombinant DNA biology.protein Electrophoresis Polyacrylamide Gel Glycoprotein Baculoviridae |
| Zdroj: | Gene. 134:75-81 |
| ISSN: | 0378-1119 |
| DOI: | 10.1016/0378-1119(93)90176-4 |
| Popis: | 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 HgCl 2 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. |
| Databáze: | OpenAIRE |
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