Recombinant Expression of Pleurocidin cDNA Usingthe <i>Pichia pastoris</i> Expression System

Abstract

This research utilized the Pichia pastoris expression system for recombinant expression of cDNA of pleurocidin, a small (2.7 kd) antimicrobial peptide isolated from winter flounder (Pleuronectes americanus). The Pichia vector contains the alcohol oxidase gene promoter (AOX 1), which under the induction of methanol allows for expression of heterologous protein gene inserted downstream in the vector. Two strains of P pastoris were used as host cells, the wild type (P pastoris X-33((mut(+)))) and the mutant (P pasatoris KM71H((mut(s))) ). Polymerase chain reaction (PCR) and DNA sequencing showed that pleurocidin cDNA was successfully integrated into the P pastoris genome. Reverse transcription (RT)-PCR showed that pleurocidin was transcribed by both Pichia host strains. Affinity chromatography, SDS-PAGE, and immunological techniques were used for purification and detection of recombinant peptide. Although there was strong evidence of transcription of pleurocidin cDNA, the Pichia system requires further optimization to obtain detectable levels of this small peptide.