Expression of rat liver tryptophan 2,3-dioxygenase in Escherichia coli: structural and functional characterization of the purified enzyme.
The hepatic hemoprotein tryptophan 2,3-dioxygenase (TDO) is the key regulatory enzyme that, through irreversible degradation, controls the flux of tryptophan through physiologically relevant pathways. This enzyme is composed of four identical subunits and in its fully assembled tetrameric form requires 2 mol of heme (Fe(+2)-protoporphyrin IX)/mol of protein for functional competence. Using a full-length cDNA for the rat liver TDO subunit (pUC119/TDO) as the template, TDO cDNA was amplified by polymerase chain reaction (PCR) and incorporated into the expression vector pTrc99A after introduction of convenient restriction sites as well as modification of the second codon AGT to GCT to optimize its bacterial expression. DH5 alpha F' strain Escherichia coli cells transfected with this pTrc99A/TDO construct expressed soluble, functionally active, tetrameric TDO protein in high yields. The enzyme was isolated from 30,000g supernatant of cell lysates, purified by ion-exchange chromatography, and its spectral and catalytic properties were assessed in terms of its substrate and prosthetic moiety specificities. In almost all aspects, the bacterially expressed enzyme was found to be identical to that of the rat liver. Heterologous expression of the fully functional enzyme, we trust, will enable future elucidation of its structure-function relationships.
Ren
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Department of Cellular and Molecular Pharmacology, University of California, San Francisco 94143, USA.
Liu
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Licad
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Correia
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MA
D000622:Aminolevulinic Acid / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D006427:Hemin / Q000737:chemistry; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity; D014366:Tryptophan Oxygenase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D006427:Hemin; D000622:Aminolevulinic Acid; D014366:Tryptophan Oxygenase