A simple and reliable method for rapid production and purification of Pseudomonas aeruginosa haemolytic phospholipase C

Abstract

Aims: To design a simple method to produce active recombinant Pseudomonas aeruginosa haemolytic phospholipase C (PLC). Method and Results: Pseudomonas aeruginosa PLC is a virulence factor mainly involved in inflammatory and cytotoxic responses. While ammonium sulphate purification requires large amounts of bacterial suspensions and leads to low yields, production of recombinant protein in Escherichia coli is no more successful because of frequent inclusion bodies and accumulation of inactive PLC in the periplasmic space. Using an inducible system based on the glucose-repressed inv1 promoter in the yeast Schizosaccharomyces pombe, we were able to produce up to 10 IU ml )1 of pure toxin within 24 h. Conclusions: 1This work describes the first method to easily get recombinant haemolytic PLC. Significance and Impact of the Study: This method provides a powerful tool to study the mechanisms leading to its cellular toxicity.