ABSTRACT
A six-His peptide was genetically engineered to the C-terminus of Agrobacterium radiobacter N-carbamoyl-D-amino acid amidohydrolase monomer to facilitate the protein purification with immobilized metal affinity chromatography (IMAC). The fusion enzyme, named as DCaseH, was overexpressed in Escherichia coli and one-step IMAC-purified. The production study showed that DCaseH was optimally produced at 15 degrees C for 25 h by the induction of 0.05 mM IPTG. Both Co(2+)-chelated TANOL gels and Ni(2+)-chelated nitriloacetic acid agarose gels efficiently purified DCaseH, with the former yielding purer enzyme than the latter. Highly pure DCaseH was obtained in the former purification with the addition of 5 mM imidazole in the washing buffer, and the specific enzyme activity was increased more than 11-fold. Denaturing IMAC purification successfully purified DCaseH from inclusion bodies that were mostly composed of the overexpressed DCaseH, while the attempt to refold the purified enzyme by either dialysis or solid-state refolding was not achieved. The purified native enzyme was optimally active at pH 6.5 and 50 degrees C, and the presence of 10% glycerol increased the activity. The molecular modeling of dimeric DCaseH indicated that the six-His tags were freely exposed to the protein surface, resulting in the selective and effective IMAC purification of DCaseH.
