Viral Hepatitis
Copyright ©2005 Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. May 28, 2005; 11(20): 3060-3064
Published online May 28, 2005. doi: 10.3748/wjg.v11.i20.3060
Expression and purification of the complete PreS region of hepatitis B Virus
Qiang Deng, Yu-Ying Kong, You-Hua Xie, Yuan Wang
Qiang Deng, Yu-Ying Kong, You-Hua Xie, Yuan Wang, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes of Life Science, Chinese Academy of Sciences, Shanghai 200031, China
Qiang Deng, You-Hua Xie, Yuan Wang, Sino-France Center for Life Science and Genome Research, Shanghai 200031, China
Author contributions: All authors contributed equally to the work.
Supported by the Basic Research Program from Ministry of Science and Technology of China, No. G1999054105, and special funds for Sino-France Center for Life Science and Genome Research from Chinese Academy of Sciences and Pasteur Institute in France
Correspondence to: Yuan Wang, 320 Yue-Yang Road, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China.
Telephone: +86-21-54921103 Fax: +86-21-54921011
Received: May 29, 2004
Revised: May 30, 2004
Accepted: July 11, 2004
Published online: May 28, 2005

AIM: To express the complete PreS region of HBV in E.coli with good solubility and stability, and to establish an effective method for purification of the recombinant PreS protein.

METHODS: The complete PreS region (PreS1 and PreS2) was fused into a series of tags including glutathione S-transferase (GST), dihydrofolate reductase (DHFR), maltose binding protein (MBP), 6×histidine, chitin binding domain (CBD), and thioredoxin, respectively. Expression of recombinant PreS fusion proteins was examined by SDS-PAGE analysis and confirmed by Western blot. Two fusion proteins, thio-PreS, and PreS-CBD, with desirable solubility and stability, were subjected to affinity purification and further characterization.

RESULTS: Recombinant PreS fusion proteins could be synthesized with good yields in E.coli. However, most of these proteins except for thio-PreS and PreS-CBD were vulnerable to degradation or insoluble as revealed by SDS-PAGE and Western blot. Thio-PreS could be purified by affinity chromatography with nickel-chelating sepharose as the matrix. However, some impurities were also co-purified. A simple freeze-thaw treatment yielded most of the thio-PreS proteins in solution while the impurities were in the precipitate. Purified thio-PreS protein was capable of inhibiting the binding of HBV virion to a specific monoclonal antibody against an epitope within the PreS1 domain.

CONCLUSION: Increased solubility and stability of the complete PreS region synthesized in E.coli can be achieved by fusion with the thioredoxin or the CBD tag. A simple yet highly effective method has been established for the purification of the thio-PreS protein. Purified thio-PreS protein likely assumes a native conformation, which makes it an ideal candidate for studying the structure of the PreS region as well as for screening antivirals.

Keywords: Hepatitis B virus, PreS, Expression, Purification