Basic Research
Copyright ©2005 Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jan 7, 2005; 11(1): 118-121
Published online Jan 7, 2005. doi: 10.3748/wjg.v11.i1.118
Heterologous expression of active human uridine diphosphate glucuronosyltransferase 1A3 in Chinese hamster lung cells
Ya-Kun Chen, Xin Li, Shu-Qing Chen, Su Zeng
Ya-Kun Chen, Xin Li, Shu-Qing Chen, Su Zeng, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310031, Zhejiang Province, China
Author contributions: All authors contributed equally to the work.
Supported by the National Natural Science Foundation of China, No. C30100232 to Xin Li, No. C30225047 to Su Zeng, and the Zhejiang Provincial Natural Science Foundation, No. C300487 to Xin Li
Correspondence to: Assistant professor Xin Li, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310031, Zhejiang Province, China. lixin@uams.edu
Telephone: +86-571-87217406
Received: April 7, 2004
Revised: April 8, 2004
Accepted: May 13, 2004
Published online: January 7, 2005
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-HPLC) coupled with a diode array detector (DAD). The quercetin glucuronide was confirmed by hydrolysis with β-glucuronidase. Control experiments were performed in parallel. The transcriptions of recombinants were also determined by RT-PCR.

RESULTS: The gene was confirmed to be an allele (UGT1A3-3) of UGT1A3 by DNA sequencing. The fragment was introduced into pcDNA3.1 (+) successfully. Several colonies were obtained under the selection pressure of G418. The result of RT-PCR showed transcription of recombinants in mRNA level. Glucuronidation assay and HPLC analysis indicated UGT1A3 expressed heterologously in CHL cells was in an active form, and one of the gulcuronides corresponding to quercetin was also detected.

CONCLUSION: Correct sequence of UGT1A3 gene can be obtained, and active UGT1A3 enzyme is expressed heterologously in CHL cells.

Keywords: Uridine Diphosphate Glucuronosyltransferase 1A3; Lung