ISOLATION OF THE PICHIA-PASTORIS GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE GENE AND REGULATION AND USE OF ITS PROMOTER

We report the cloning and sequence of the glyceraldehyde-3-phosphate d ehydrogenase gene (GAP) from the yeast Pichia pastoris. The gene is pr edicted to encode a 35.4-kDa protein with significant sequence similar ity to glyceraldehyde-3-phosphate dehydrogenases from other organisms. Promoter studies in P. pastoris using bacterial beta-lactamase as a r eporter showed that the GAP promoter (P-GAP) is constitutively express ed, although its strength varies depending on the carbon source used f or cell growth. Expression of beta-lactamase under control of P-GAP in glucose-grown cells was significantly higher than under control of th e commonly employed alcohol oxidase 1 promoter (P-AOX1) in methanol-gr own cells. As an example of the use of P-GAP, we showed that beta-lact amase synthesized under transcriptional control of P-GAP is correctly targeted to peroxisomes by addition of either a carboxy-terminal or an amino-terminal peroxisomal targeting signal. P-GAP has been successfu lly utilized for synthesis of heterologous proteins from bacterial, ye ast, insect and mammalian origins, and therefore is an attractive alte rnative to P-AOX1 in P. pastoris.