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(muts)). 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.
Highlights
Pleurocidin is a 25-amino-acid peptide isolated from the skin mucus secretions of the winter flounder (Pleuronectes americanus) (Cole et al [1])
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)
Polymerase chain reaction (PCR) and DNA sequencing showed that pleurocidin cDNA was successfully integrated into the P pastoris genome
Summary
Pleurocidin is a 25-amino-acid peptide isolated from the skin mucus secretions of the winter flounder (Pleuronectes americanus) (Cole et al [1]). Pleurocidin has been found to be effective against clinical isolates from patients with urinary and respiratory tract infections and hip and bone wounds, and acts synergistically with D-cycloserine against the food-related and medically significant Mycobacterium tuberculosis (Cole et al [3]). These findings suggest that pleurocidin has potential application in aquaculture, as well as for food and pharmaceutical applications. The high cost of chemically synthesizing pleurocidin or isolating it directly from the winter flounder is prohibitive for the large-scale applications. Producing the peptide in a microbiological system through the use of a genetically modified organism would allow for large-scale production and purification
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