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Genetics and Molecular Biology |
Service de Dermatologie (DHURDV), Laboratoire de Mycologie, BT422, Centre Hospitalier Universitaire Vaudois, 1011 Lausanne, Switzerland1
Department of Medical Microbiology, Institute of Hygiene, University of Göttingen, Germany2
Department of Medical and Organic Chemistry, School of Pharmacy, University of Wisconsin, Madison, USA3
Author for correspondence: M. Monod. Tel: +41 21 314 0376. Fax: +41 21 314 0378. e-mail: Michel.Monod{at}chuv.hospvd.ch
Pathogenic yeasts of the genus Candida secrete aspartic proteinases (Sap) which are synthesized as preproenzymes. Expression of the C. albicans SAP1 gene lacking the propeptide-coding region in the methylotrophic yeast Pichia pastoris does not lead to the secretion of the enzyme into the culture supernatant, but results in an accumulation of recombinant protein in the cell. Co-expression in this system of the unattached propeptide from Sap1p, as well as from other Saps, restored Sap1p secretion. A deletion analysis revealed that only a 12 aa sequence in the propeptide, corresponding to a highly conserved region in all Sap propeptides, was necessary and sufficient to produce a large amount of Sap1p in culture supernatant. No Sap1p was secreted when Sap1p was produced with a propeptide carrying an F to D mutation in the identified 12 aa sequence. However, the simultaneous production of equivalent amounts of Sap1p and His-tagged Sap1p (H6-Sap1p) with a mutated and a non-mutated propeptide, respectively, led to the secretion of both proteins in a ratio of approximately 1:2. The restoration of Sap1p secretion occurred at the expense of secretion of H6-Sap1p since the total activity was comparable to that of strains producing only H6-Sap1p with a non-mutated propeptide. In contrast, the proteolytic activity of strains secreting Sap1p and H6-Sap1p both with a functional propeptide was twice that of strains producing either Sap1p or H6-Sap1p alone, and the two enzymes were found in an equivalent amount in the culture supernatant. Altogether, these results show that the propeptide can only function once and that the maturation of recombinant C. albicans secreted aspartic proteinase Sap1p is directed through a combination of intra- and inter-molecular pathways.
Keywords: SAP1 gene, protein secretion, signal peptides, recombinant proteins, Pichia pastoris
Abbreviations: Sap, secreted aspartic proteinase; H6-Sap1p, His6-tagged Sap1p
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