Expression, Purification, and Characterization of Rat Aromaticl-Amino Acid Decarboxylase inEscherichia coli

Abstract

A cDNA encoding rat aromaticl-amino acid decarboxylase (AADC) was successfully expressed inEscherichia coliusing a T7 RNA polymerase expression system. Two types of expression vectors were tested and revealed to be equivalent to produce AADC. The enzyme was purified in both cases. The ratio of recovery of the pure active recombinant protein was better when the purification of the protein was made easier by addition of a short His–Tag at the C-terminal moiety of AADC, as achieved in the case of pET-20b+ vector expression. Spectral characteristics of the bound pyridoxal-5′-phosphate were essentially identical to the spectral properties of rat AADC. Kinetic constantsKmandVmaxof recombinant AADC for the natural substratesl-dihydroxyphenylalanine and 5-hydroxytryptamine were 0.14 mmand 8444 U/mg, and 0.066 mmand 1813 U/mg, respectively. These values were in good agreement with previously reported values for AADC of the rat and other mammalian species.