Production, IMAC purification, and molecular modeling of N-carbamoyl-D-amino acid amidohydrolase C-terminally fused with a six-his peptide.

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