| 1 | Expression and purification of the complete PreS region of hepatitis B Virus显示文摘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 Stransferase (GST), dihydrofolate reductase (DHFR), maltose binding protein (MBP), 6x 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 besynthesized 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 copurified. 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 achievedby 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. | QiangDeng Yu-YingKong You-HuaXie YuanWang | 2005 | World Journal of Gastroenterology2005，11，20： | 8 |
| 2 | Screening for PreS specific binding ligands with a phage displayed peptides library显示文摘AIM: To construct a random peptide phage display library and search for peptides that specifically bind to the PreS region of hepatitis B virus (HBV).METHODS: A phage display vector, pFuse8, based on the gene 8 product (pⅧ) of M13 phage was made and used to construct a random peptide library. E. coli derived thioredoxin-PreS was purified with Thio-bond beads, and exploited as the bait protein for library screening. Five rounds of bio-panning were performed. The PreS-binding specificities of enriched phages were characterized with phage ELISA assay.RESULTS: A phage display vector was successfully constructed as demonstrated to present a pⅧ fused HBV PreS1 epitope on the phage surface with a high efficiency.A cysteine confined random peptide library was constructed containing independent clones exceeding 5±108 clone forming unit (CFU). A pool of phages showing a PreS-binding specificity was obtained after the screening against thioPres with an enrichment of approximately 400 times. Five phages with high PreS-binding specificities were selected and characterized. Sequences of the peptides displayed on these phages were determined.CONCLUSION: A phage library has been constructed,with random peptides displaying as pⅧ-fusion proteins.Specific PreS-binding peptides have been obtained, which may be useful for developing antivirals against HBV infection. | QiangDeng MingZhuang Yu-YingKong You-HuaXie YuanWang | 2005 | World Journal of Gastroenterology2005，11，26： | 4 |
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