Expression, reactivation, and purification of enzymes from Haloferax volcanii in Escherichia coli

Enzymes from extreme halophiles have potential as catalysts in biotransform ations. We have developed methods for the expression in Escherichia coli an d purification of two enzymes from Haloferax volcanii: dihydrolipoamide deh ydrogenase and citrate synthase. Both enzymes were expressed in E. coli usi ng the cytoplasmic expression vectors, pET3a and pET3d. Citrate synthase wa s soluble and inactive, whereas dihydrolipoamide dehydrogenase was expresse d as inclusion bodies. Citrate synthase was reactivated following overnight incubation in 2 M KCl, and dihydrolipoamide dehydrogenase was refolded by solubilisation in 8 M urea followed by dilution into a buffer containing 2 M KCl, 10 mu M FAD, 1 mM NAD, and 0.3 mM GSSG/3 mM GSH. Maximal activity wa s obtained after 3 days incubation at 4 degrees C. Purification of the two active enzymes was carried out using high-resolution methods. Dihydrolipoam ide dehydrogenase was purified using copper-based metal-ion affinity chroma tography in the presence of 2 M KCl. Citrate synthase was recovered using d ye-affinity chromatography in the presence of salt. A high yield of active enzyme was obtained in both cases. Following purification, characterisation of both recombinant proteins showed that their kinetics and salt-dependenc e were comparable to those of the native enzymes. Expression of active prot ein was attempted both by growth of E. coil in the presence of salt and bet aine, and also by using periplasmic expression vectors in combination with a high salt growth media. Neither strategy was successful. (C) 1999 John Wi ley & Sons, Inc.