title: Purification and characterization of a catalytically active human liver UDP-glucuronosyltransferase expressed as a fusion protein in E. coli.
abstract: The purification and the characterization of functional human liver UDP-glucuronosyltransferase 2B4 produced as a Staphylococcus aureus protein A fusion protein in E. coli are described. The purified fusion protein was able to catalyze the glucuronidation of hyodeoxycholic acid, the major substrate described for this isoform to date. The effects of the amount and the nature of the phospholipids upon reconstitution into phospholipid micelles were investigated. Apparent determined Km values for hyodeoxycholic acid and UDP-glucuronic acid were 0.55 and 0.43 mM, respectively. Moreover, photoaffinity labelling of the fusion protein with a photoactivatable analog of UDP-glucuronic acid strongly suggested that this recombinant protein exhibited similar binding properties as the microsomal protein, which emphasizes its use for further structural analyses.
authors.affiliation: Centre du Médicament, URA CNRS 597, Nancy, France.
authors.forename: T
authors.initials: T
authors.lastname: Pillot
authors.forename: M
authors.initials: M
authors.lastname: Ouzzine
authors.forename: S
authors.initials: S
authors.lastname: Fournel-Gigleux
authors.forename: C
authors.initials: C
authors.lastname: Lafaurie
authors.forename: A
authors.initials: A
authors.lastname: Radominska
authors.forename: R
authors.initials: R
authors.lastname: Lester
authors.forename: R
authors.initials: R
authors.lastname: Drake
authors.forename: S
authors.initials: S
authors.lastname: Treat
authors.forename: G
authors.initials: G
authors.lastname: Siest
authors.forename: J
authors.initials: J
authors.lastname: Magdalou
mesh_terms: D000345:Affinity Labels; D003840:Deoxycholic Acid / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D014453:Glucuronosyltransferase / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008823:Micelles; D010743:Phospholipids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
chemical_list: D000345:Affinity Labels; D008823:Micelles; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; D003840:Deoxycholic Acid; C010471:hyodeoxycholic acid; D014453:Glucuronosyltransferase