Li-Xin Zhu

Expression, purification, immunological characterization and application of Escherichia coli-derived hepatitis C virus E2 proteins

The envelope glycoprotein E2 of hepatitis C virus (HCV) has been shown to bind human target cells. Anti‐E2 antibodies have been associated with both recovery from natural infection in humans and protection from challenge with homologous HCV in chimpanzees. Therefore E2 has become a major target for the development of anti‐HCV vaccines. Two E2 fragments [amino acids (aa) 450–565 and aa 385–565] derived from a subtype 1b HCV genome were expressed as N‐terminally hexahistidine‐tagged proteins in Escherichia coli and purified to over 85% purity. Both proteins were specifically recognized by homologous hepatitis‐C‐patients serum on Western blotting, suggesting that these E. coli‐derived E2 proteins displayed E2‐specific antigenicity. E2‐116 (aa 450–565) elicited strong antibody responses in BALB/c mice and rabbits. Rabbit antiserum raised against renatured E2‐116 (RE2–116R) was able to recognize subtype 1b and 1a E2 glycoproteins expressed in mammalian cells on Western blotting. E2‐181 (aa 385–565) reacted with 40% of anti‐HCV+ patients’ sera in ELISA. RE2‐116R and E2‐181 were successfully used in preliminary modified vaccinia virus Ankara‐ and DNA‐based E2 vaccine studies for detecting antigen expression in vitro and assessing induced humoral immune responses in mice. The E2 proteins and rabbit antiserum reported here could find wider applications in the development of effective diagnostic, prophylactic and therapeutic measures against HCV.

High-level expression of the C-terminal hydrophobic region of HCV E2 protein ectodomain in E-coli

High-level expression of hepatitis C virus (HCV) E2 protein fragments encompassing its C-terminal hydrophobic region (downstream of aa 661) in Escherichia coli has been proven difficult. The extreme hydrophobicity of this region has been suspected to be detrimental to the host. In this work, we found that the C-terminal region (downstream of aa 565) of E2 ectodomain interfered with full-length expression of E2 fragments in E. coli. Nonetheless, when the central region (aa 484–622) of E2 was deleted, full-length protein was efficiently produced. C-terminal region aa 567–700 could not be efficiently expressed individually or as mouse dihydrofolate reductase (DHFR) fusion protein. However, a mutant that emerged in the cloning process was able to express full-length DHFR fusion protein. Sequencing analysis reveals the mutation to be a short frame-shift around the fusion junction, altering aa 568–571 of E2. C-terminal region of E2 ectodomain (aa 567–730) carrying this mutation was successfully expressed as hexa-histidine-tagged protein to a high level. The protein was highly insoluble and was purified under denaturing conditions. The purified protein displayed HCV E2-specific antigenicity in Western blot and specific rabbit antiserum was raised against it. These results demonstrate that hydrophobicity of the C-terminal region of E2 ectodomain is not harmful to E. coli host and has no dominative adverse effect on its bacterial expression. Other nucleotide and/or amino acid sequence properties seem to play a more important role. This finding opens up new possibilities for the development of novel bacterially-derived E2 proteins for research and clinical applications.