CLONING AND CHARACTERIZATION OF THE PICHIA-PASTORIS PRC1 GENE ENCODING CARBOXYPEPTIDASE-Y

We purified a 58 kDa serine protease from culture-supernatant of Pichi a pastoris and found that the NH2-terminal amino acid sequence of this protease is closely homologous to that of mature protein of Saccharom yces cerevisiae carboxypeptidase Y (CPY), which is encoded by the PRC1 gene. Using the S. cerevisiae PRC1 gene as a hybridization probe, a c ross-hybridizing fragment of P. pastoris genomic DNA was identified an d the gene, PRC1, encoding CPY, was cloned. The open reading frame of the P. pastoris PRC1 gene consists of 1569 bp encoding a protein of 52 3 amino acids. The molecular mass of the protein is calculated to be 5 9.44 kDa without sugar chains. The protein comprises 20 amino acids of pre (signal)-peptide, 87 amino acids of pro-peptide and 416 amino aci ds of mature peptide, and has four N-glycosylation sites. The NH2-term inal amino acid sequence of mature peptide is completely identical wit h that of the protease purified from the culture-supernatant. There is 61% identity between the amino acid sequences of P. pastoris Prc1p an d S. cerevisiae Prc1p. Chromosomal disruption of the PRC1 gene resulte d in the loss of CPY activity. Over-expression of the PRC1 gene under regulation of the P. pastoris AOX1 promoter resulted in accumulation o f a large amount of active CPY in the intracellular fraction, and secr etion of a slightly larger molecule that is thought to be pro-CPY. The nucleotide sequence data reported in this paper will appear in the EM BL Nucleotide Sequence Databases under the Accession Number X87987.