Heterologous expression of active human uridine diphosphate glucuronosyltransferase 1A3 in Chinese hamster lung cells.

ABSTRACT:

Aim

To obtain the active human recombinant uridine diphosphate glucuronosyltransferase 1A3 (UGT1A3) enzyme from Chinese hamster lung (CHL) cells.

Methods

The full-length UGT1A3 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using total RNA from human liver as template. The correct fragment confirmed by sequencing was subcloned into the mammalian expression vector pcDNA3.1 (+), and the recombinant vector was transfected into CHL cells using a calcium phosphate method. Expressed UGT1A3 protein was prepared from CHL cells resistant to neomycin (G418). Then the protein was added into a reaction mixture for glucuronidation of quercetin. The glucuronidation activity of UGT1A3 was determined by reverse phase-high performance liquid chromatography (RP

SUBMITTER: Chen YK 

PROVIDER: S-EPMC4205369 | biostudies-literature | 2005 Jan

REPOSITORIES: biostudies-literature