Sy. Ren et al., EXPRESSION OF RAT-LIVER TRYPTOPHAN 2,3-DIOXYGENASE IN ESCHERICHIA-COLI - STRUCTURAL AND FUNCTIONAL-CHARACTERIZATION OF THE PURIFIED ENZYME, Archives of biochemistry and biophysics, 333(1), 1996, pp. 96-102
The hepatic hemoprotein tryptophan 2,3-dioxygenase (TDO) is the key re
gulatory enzyme that, through irreversible degradation, controls the f
lux of tryptophan through physiologically relevant pathways. This enzy
me is composed of four identical subunits and in its fully assembled t
etrameric form requires 2 mol of heme (Fe+2-protoporphyrin IX)/mol of
protein for functional competence. Using a full-length cDNA for the ra
t liver TDO subunit (pUC119/TDO) as the template, TDO cDNA was amplifi
ed by polymerase chain reaction (PCR) and incorporated into the expres
sion vector pTrc99A after introduction of convenient restriction sites
as well as modification of the second codon AGT to GCT to optimize it
s bacterial expression. DH5 alpha F' strain Escherichia coli cells tra
nsfected with this pTrc99A/TDO construct expressed soluble, functional
ly active, tetrameric TDO protein in high yields. The enzyme was isola
ted from 30,000g supernatant of cellysates, purified by ion-exchange c
hromatography, and its spectral and catalytic properties were assessed
in terms of its substrate and prosthetic moiety specificities, in alm
ost all aspects, the bacterially expressed enzyme was found to be iden
tical to that of the rat liver. Heterologous expression of the fully f
unctional enzyme, we trust, will enable future elucidation of its stru
cture-function relationships. (C) 1996 Academic Press, Inc.