Cloning and characterization of the Pichia pastoris PRC1 gene encoding carboxypeptidase Y

✍ Scribed by Ohi, Hideyuki; Ohtani, Wataru; Okazaki, Noriko; Furuhata, Naoto; Ohmura, Takao

• Publisher: John Wiley and Sons
• Year: 1996
• Tongue: English
• Weight: 893 KB
• Volume: 12
• Category: Article
• ISSN: 0749-503X
• DOI: 10.1002/(sici)1097-0061(199601)12:1<31::aid-yea877>3.0.co;2-s

✦ Synopsis

We purified a 58 kDa serine protease from culture-supernatant of Pichia pastoris and found that the NH,-terminal amino acid sequence of this protease is closely homologous to that of mature protein of Saccharomyces cerevisiae carboxypeptidase Y (CPY), which is encoded by the PRCl gene. Using the S. cerevisiae PRCl gene as a hybridization probe, a cross-hybridizing fragment o f P. pastoris genomic DNA was identified and the gene, PRCl, encoding CPY, was cloned. The open reading frame of the P. pastoris PRCl gene consists of 1569 bp encoding a protein of 523 amino acids. The molecular mass of the protein is calculated to be 59.44 kDa without sugar chains. The protein comprises 20 amino acids ofpre (signal)-peptide, 87 amino acids ofpro-peptide and 416 amino acids of mature peptide, and has four N-glycosylation sites. The NH,-terminal amino acid sequence of mature peptide is completely identical with that of the protease purified from the culture-supernatant. There is 61% identity between the amino acid sequences of P. pastoris Prclp and S. cerevisiae Prclp. Chromosomal disruption o f the PRCl gene resulted in the loss of CPY activity. Over-expression of the PRCl gene under regulation of the P. pastoris AOXl promoter resulted in accumulation of a large amount of active CPY in the intracellular fraction, and secretion of a slightly larger molecule that is thought to be pro-CPY.