title: Expression, purification, and characterization of rat aromatic L-amino acid decarboxylase in Escherichia coli.
abstract: A cDNA encoding rat aromatic L-amino acid decarboxylase (AADC) was successfully expressed in Escherichia coli using 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 constants Km and Vmax of recombinant AADC for the natural substrates L-dihydroxyphenylalanine and 5-hydroxytryptamine were 0.14 mM and 8444 U/mg, and 0.066 mM and 1813 U/mg, respectively. These values were in good agreement with previously reported values for AADC of the rat and other mammalian species.
authors.affiliation: Laboratoire de Biochimie cellulaire, Collège de France, 11 place Marcelin Berthelot, Paris Cedex 05, 75231, France.
authors.forename: F
authors.initials: F
authors.lastname: Jebai
authors.forename: N
authors.initials: N
authors.lastname: Hanoun
authors.forename: M
authors.initials: M
authors.lastname: Hamon
authors.forename: J
authors.initials: J
authors.lastname: Thibault
authors.forename: G
authors.initials: G
authors.lastname: Peltre
authors.forename: F
authors.initials: F
authors.lastname: Gros
authors.forename: M
authors.initials: M
authors.lastname: Krieger
mesh_terms: D006916:5-Hydroxytryptophan / Q000378:metabolism; D000818:Animals; D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000302:isolation & purification* / Q000378:metabolism; D015153:Blotting, Western; D004295:Dihydroxyphenylalanine / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D007525:Isoelectric Focusing; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D004295:Dihydroxyphenylalanine; D006916:5-Hydroxytryptophan; D001142:Aromatic-L-Amino-Acid Decarboxylases