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Title 

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

Authors 

Sun Boon RhyunByung Rae JinHeung Rok ParkHyo Jeong Hong

Publisher 

Elsevier

Issue Date 

1994

Citation 

Journal of Biotechnology, vol. 36, no. 0, pp. 221-230

Keywords 

Anion-exchange chromatographyE. coli expressionFactor Xa digestionGel filtrationHepatitis B virus (HBV) preS1 peptideMBP fusion proteinhybrid proteinmaltose binding proteinenzyme linked immunosorbent assaygene expression

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.

ISSN 

0168-1656

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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