Expression of Hepatitis C Virus E2 Ectodomain in <italic>E. coli</italic> and Its Application in the Detection of Anti-E2 Antibodies in Human Sera

Abstract

The second envelope glycoprotein (E2) of hepatitis C virus has been shown to bind human target cells and has become a major target for the development of anti-HCV vaccines. Anti-E2 antibodies have been suggested to be of clinical significance in diagnosis, treatment and prognosis of hepatitis C. However, large-scale expression and purification of E2 proteins in mammalian cells is difficult. As an alternative, E2 fragment (aa 385 C730) with a four-amino-acid mutation (aa 568 C571 PCNI to RVTS) was expressed as hexa-histidine-tagged full length protein [E2N730(m)] in E. coli and purified to over 85% purity. Purified E2N730(m) was specifically recognized by homologous hepatitis C patient serum in Western blot, suggesting that it displayed E2-specific antigenicity. Rabbit antiserum raised against E2N730(m) recognized E2 glycoproteins expressed in mammalian cells in Western blot. Purified E2N730(m) was used to detect anti-E2 antibodies in human sera and showed better specificity and sensitivity than previously reported C-terminally truncated E2 fragment (aa 385 C565). Association between anti-E2 antibodies in patient sera and HCV RNA status was also demonstrated using this E. coli-derived protein. E2N730(m) might serve as an inexpensive alternative to mammalian cell-expressed E2 proteins in clinical and research applications.

Generating Recombinant Antibodies to Membrane Proteins through Phage Display

“…One approach to circumvent this bottleneck is to express their extracellular domains ( i.e. , ectodomains) as soluble and secreted fusion proteins (Figure 2A) in mammalian [24,25,26,27], bacterial [28,29,30,31], yeast [32,33] and insect cells [27,34]. For example, as long as the fusion partner does not interfere with proper folding of the ectodomains, they can be expressed fused to the Fc region of IgG or an affinity tag, such as the His 6 -tag [35] and FLAG tag [36].…”