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Expression and purification of the complete PreS region of hepatitis B Virus.
- Source :
-
World journal of gastroenterology [World J Gastroenterol] 2005 May 28; Vol. 11 (20), pp. 3060-4. - Publication Year :
- 2005
-
Abstract
- 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.<br />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), 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.<br />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.<br />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.
- Subjects :
- Escherichia coli metabolism
Hepatitis B Surface Antigens biosynthesis
Hepatitis B Surface Antigens isolation & purification
Hepatitis B virus physiology
Protein Precursors biosynthesis
Protein Precursors isolation & purification
Recombinant Proteins biosynthesis
Recombinant Proteins isolation & purification
Viral Envelope Proteins biosynthesis
Viral Envelope Proteins isolation & purification
Subjects
Details
- Language :
- English
- ISSN :
- 1007-9327
- Volume :
- 11
- Issue :
- 20
- Database :
- MEDLINE
- Journal :
- World journal of gastroenterology
- Publication Type :
- Academic Journal
- Accession number :
- 15918190
- Full Text :
- https://doi.org/10.3748/wjg.v11.i20.3060