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-polyhydroxyalkanoate (mcl-PHA) polymerase was developed. This process is based on solubilization and activation of inactive polymerase inclusion bodies by incubation 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-exchange resin in the presence of dithiothreitol and glycerol, the mcl-PHA polymerase could be extracted in an active and soluble form with a final yield of 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 components, suggesting its potential application for production of biopolymer. The procedure used here may be of general value in solubilizing and activating purified inactive labile enzymes.
Ren
Q
Q
Institute of Biotechnology, ETH Hönggerberg, CH-8093 Zürich, Switzerland.
De Roo G
Kessler
B
B
Witholt
B
B
D000217:Acyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001426:Bacterial Proteins; D002852:Chromatography, Ion Exchange / Q000379:methods; D002479:Inclusion Bodies / Q000201:enzymology*; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification; D012995:Solubility
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D000217:Acyltransferases; C067032:poly(3-hydroxyalkenoate)polymerase