Recombinant Expression of Pleurocidin cDNA UsingthePichia pastorisExpression System

[摘要] This research utilized thePichia pastorisexpressionsystem for recombinant expression of cDNA of pleurocidin, a small(2.7kd) antimicrobial peptide isolated from winter flounder (Pleuronectes americanus). ThePichiavector contains the alcohol oxidase gene promoter (AOX 1), which under the induction of methanol allows for expression of heterologousprotein gene inserted downstream in the vector. Two strains ofP pastoriswere used as host cells, the wild type(P pastorisX-33a(mut+)) and the mutant (P pasatorisKM71Ha(muts)). Polymerase chain reaction (PCR) and DNA sequencing showed that pleurocidin cDNA was successfully integrated into theP pastorisgenome.Reverse transcription (RT)-PCR showed that pleurocidin was transcribed by bothPichiahost 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,thePichiasystem requires further optimization to obtaindetectable levels of this small peptide.

[学科分类] 基础医学