Cloning and Expression of a Novel PHO85 Associated Protein PAP1 Gene of Saccharomyces cerevisiae

Yeast regulatory factor PHO85, which is a cyclin-dependent kinase (DCK), participates in the regulation of the cell cycle and the expression of the acid phosphatase gene. Using PHO85 as target, we have cloned from the yeast two-hybrid genomic library a novel gene (PAP1) with product which can asscociate with the PHO85 protein. The PAP1 gene including the 5apo; and 3apo; flanking sequence was cloned and sequenced. It codes for a protein composed of 284 amino acids, which contains a cyclin conservative region (150-263 aa), and shares 63% similarity with the conservative region of PHO80. The N-terminal of the PAP1 protein abounds in PEST sequence, which is believed to be common to rapidly degraded proteins. Using the yeast two-hybrid system, we demonstrated that both the full-length PAP1 protein and the C-terminal (99-285 aa) can interact with PHO85. The coding sequence of the PAP1 gene was cloned and sequenced, then arranged under the control of the P(L) promoter, and expressed in E. coli BL21 (DE3) pLysS. After heat induction at 42 degrees, protein band of approximately 32 kD was observed in the SDS-PAGE, which fits the molecular size of the hypothetical PAP1 protein. The 10 N-terminal amino acids of the 32 kD protein agreed with the deduced PAP1 amino acid sequence. The protein was found as inclusion body in E. coli, and accounted for 50% of the precipitant after purification.