Recovery of active medium-chain-length-poly-3-hydroxyalkanoate polymerase from inactive inclusion bodies using ion-exchange resin

A novel process for the purification of active medium-chain-length-polyhydr oxyalkanoate (mcl-PHA) polymerase was developed. This process is based on s olubilization and activation of inactive polymerase inclusion bodies by inc ubation with ion-exchange resin, The mcl-PHA polymerase 1 from Pseudomonas oleovorans was overproduced from the Palk promoter. Most of the polymerase produced was sequestered in the cytoplasm as an inactive form in insoluble aggregates. By incubating the protein aggregates with S-Sepharose ion-excha nge resin in the presence of dithiothreitol and glycerol, the mcl-PHA polym erase could be extracted in an active and soluble form with a final yield o f about 5.2 mg/g of cell dry weight. The solubilized polymerase was able to catalyse the in vitro synthesis of mcl-PHA without any additional cell com ponents, suggesting its potential application for production of biopolymer. The procedure used here may be of general value in solubilizing and activa ting purified inactive labile enzymes.