Studies on the expression, purification and renaturation of recombinant N-acety-L-ornithine deacetylase

The argE gene from Escherichia coli coding for N-acety-L-ornithine deacetylase (NAOase), the key enzyme involved in the L-arginine biosynthesis, had been cloned in pET22b and transformed into BL21 (DE3). With 32.5% expression level in the optimal fermentation medium at 37°C, most NAOase was expressed as inclusion bodies. The soluble and active proportion could be slightly increased when expressed at low temperature. The specific activity of soluble NAOase purified by Ni-NTA resin chromatography was 1193.2 u/mg. The species and proportions of whole cell proteins varied with induction conditions. The inclusion bodies expressed at 37°C was more pure than 22°C after gradient wash with urea. Inclusion bodies could be partly refolding and reactivated by dilution and dialysis. Low protein concentration and suitable rate of oxidant/reducing agents were important to renaturation. In the optimal conditions 17.78% of Urea-denatured NAOase could be refolding and reactivated by dilution. The purified fusion protein was obtained after wash, solubilization and Ni-NTA resin affinity chromatography purification of inclusion bodies.