Haliotis rubra hemocyanin: sequencing, recombinant expression and investigation of the binding activities of expressed function units with HSV-1

Abstract

Hemocyanin (HrH) has been shown to possess antiviral activity against HSV-1 by selectively binding to viral glycoproteins (gB, gC, gD). Its large size however precluded the application of this protein, which required identifying smaller structure with antiherpetic ability. Aiming to this goal, the amino acid sequences and theoretical 3D structure of two isoforms that constitute HrH were determined by this study for the first time. Eight of the smallest intrinsic structure of HrH, herein called functional units (FUs), were identified in each isoform. Subsequently, the gene encoding HrH was cloned and used to establish the first recombinant bacterial expression system of HrH. All 16 FUs were expressed individually at the optimised temperature (16°C) with a glutathione S-transferase (GST) fused at the N-termini, and were exploited to reveal direct protein-protein interaction between HrH and HSV-1 glycoproteins by the GST pull-down assay. Different degrees of binding amongst three HSV-1 glycoproteins (gB>gD>gC) were found implying that the antiviral activity of HrH was partially preserved in the recombinant FUs. Amongst 16 FUs, HrH2-FUb demonstrated the most consistent binding abilities. Therefore, it was hypothesised to be the prime candidate for future study. The investigation of exploiting E. coli to fabricate HrH2-FUb by a scale-upped system was also conducted in this study. The specific affinity between glutathione and the fused GST tag was utilised to extract the GST-HrH2-FUb by affinity chromatography. Size-exclusion chromatography was employed to further purify the samples eluted from the affinity column. However, the generation of the low molecular weight GST-tagged HrH fragments revealed the limitations associated with the current bacterial system. Taken together, this study provided essential knowledge to facilitate the discovery of antiherpetic compounds from HrH, which could become novel treatment options, for HSV-1 infection in the future.