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Title: Interactions between the surface and core antigens of Hepatitis B virus
Author: Tan, Wen Siang
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1997
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The core antigen (HBcAg) of hepatitis B Virus (HBV) can be expressed in Escherichia coli where it assembles into icosahedral particles of two sizes containing 240 or 180 subunits. These particles could be fractionated by sucrose gradient centrifugation, and light scattering showed their size distribution to be essentially monodisperse. HBcAg is 183 amino acids long and highly enriched in arginine residues in the C-terminal region. Around 50% of these residues are encoded by the rare triplet AGA in E. coli. Supplementation of the level of AGA tRNA in the cell with plasmids expressing the T4 AGA tRNA gene significantly enhanced the yield of HBcAg. SDS polyacrylamide gel electrophoresis of the two kinds of particles showed that around half of their subunits were smaller than the full length HBcAg and varied in size. N-terminal sequence analysis revealed that these smaller species were heterogeneous at the extremely basic C-terminal end. In the virus, the icosahedral nucleocapsids are surrounded by an envelope consisting of cellular lipids and three related surface antigens (L-, M-, and S-HBsAg) which result from alternative translation initiation of a common reading frame. The L-HBsAg is believed to mediate the contact between the envelope and nucleocapsid. The N- and C-termini of this protein were shortened in order to define the minimum stretch of amino acids that contains the exact contact residues. To determine which residues are directly involved in the interaction, single and multiple mutations were generated by site-directed mutagenesis. The resulting mutated proteins were expressed in rabbit reticulocyte lysates, and their ability to interact with HBcAg was examined with an immunoprecipitation assay and a newly established equilibrium binding assay in solution which allows the determination of relative dissociation constants with Scratchard and non-linear regression analyses.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available