| Autor: |
Valverde, Viviane, Delmas, Pascal, Kaghad, Mourad, Loison, Gérard, Jara, Patrick |
| Zdroj: |
Journal of Biological Chemistry; June 1995, Vol. 270 Issue: 26 p15821-15826, 6p |
| Abstrakt: |
The gene encoding endothiapepsin (EAP), an extracellular aspartic proteinase from the filamentous ascomycete Cryphonectria parasitica, was expressed into Saccharomyces cerevisiae. Efficient secretion of an active and correctly processed enzyme was achieved when expressing the entire cDNA encoding prepro-EAP under the control of the galactose-inducible GRAP1yeast promoter. Since three independent, site-directed mutations of EAP, including the substitution of an aspartyl catalytic residue, resulted in the intracellular accumulation of zymogen forms, we assumed that the EAP propeptide was autocatalytically processed. As a prerequisite to further improve the specificity of EAP, we therefore attempted to bypass this self-processing step in three different ways: 1) introduction of a Kex2-like recognition site between the pro and the mature part, 2) deletion of the prosequence (pre-EAP), and 3) co-expression in transof the pre-EAP with its preprosequence. No improvement in the secretion of mutant enzymes was obtained in any of these experiments. As an alternative, we finally replaced the EAP processing site by the chymosin cleavage sequence of κ-casein. Such a modification remained efficient in directing the secretion of active EAP only when a putative α-helix structural motif was conserved at the C terminus of the pro region. |
| Databáze: |
Supplemental Index |
| Externí odkaz: |
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