High level expression of hepatitis B virus preS1 peptide in Escherichia coli

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Title
High level expression of hepatitis B virus preS1 peptide in Escherichia coli
Author(s)
Sun Boon Rhyun; Byung Rae Jin; Heung Rok Park; Hyo Jeong Hong
Bibliographic Citation
Journal of Biotechnology, vol. 36, pp. 221-230
Publication Year
1994
Abstract
PreS1 region gene fragment encoding the N-terminal 56 amino acid (aa) of hepatitis B virus (HBV, adr subtype), which encodes B- and T-cell epitopes and an hepatocyte receptor binding site, was synthesized by PCR and fused to the 3'-end of MalE gene encoding maltose-binding protein (MBP) to yield expression plasmid pMalpreS1-56. The plasmid was introduced into Escherichia coli DH5α and expressed at 37°C under the control of inducible tac promoter. The resulting fusion protein was highly expressed in a soluble form, about 40% of total cellular proteins, but it bound only partially to an amylose column. Therefore, the soluble preS1 fusion protein was purified to near homogeneity by two passages of anion-exchange chromatography followed by gel filtration. The yield of the fusion protein was 70 mg per 1 culture that had been induced by IPTG for 6 h. The purified fusion protein was specifically cleaved by a Factor Xa digestion to release the preS1 peptide, which was then purified by gel filtration to homogeneity. The purity, integrity, antigenicity and immunogenicity of the purified preS1 peptide was confirmed by glycerol-SDS-PAGE, Western analysis, N-terminal amino acid sequencing and an indirect ELISA.
Keyword
Anion-exchange chromatographyE. coli expressionFactor Xa digestionGel filtrationHepatitis B virus (HBV) preS1 peptideMBP fusion protein
ISSN
0168-1656
Publisher
Elsevier
DOI
http://dx.doi.org/10.1016/0168-1656(94)90153-8
Type
Article
Appears in Collections:
1. Journal Articles > Journal Articles
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