Expression and folding of human very-low-density lipoprotein receptor fragments: Neutralization capacity toward human rhinovirus HRV2

Minor group human rhinoviruses (HRVs) use members of the low-density lipopr otein receptor family for cell entry. To investigate the utility of recepto r fragments as viral inhibitors, various polypeptide segments derived from the ligand binding domain of human very-low-density lipoprotein receptor (V LDLR) were expressed in a soluble form in bacteria. Whereas none of the fra gments was active in virus binding immediately after recovery from the cell lysates, constructs encompassing complement type repeats 1-3, 1-6, and 1-8 spontaneously acquired virus binding activity by incubation at 4 degreesC in buffer containing Ca2+ ions and lacking any redox system. When immobiliz ed receptor-associated protein (RAP), a specific chaperone for VLDLR, was p resent during the incubation, the yield of protein active in ligand binding was substantially increased. A VLDLR fragment with repeats 4-6 failed to b ind virus; however, it bound RAP. Bacterial expression of truncated VLDLR 1 -3 at high yield, easy purification, and folding together with high inhibit ory activity toward HRV2 makes this protein a promising starting point for the development of an oligopeptide-based antiviral agent. Using sucrose den sity gradient centrifugation, we demonstrate the formation of virus-recepto r complexes. The recombinant receptors can thus be used for structure deter mination by electron cryo-microscopy. (C) 2000 Academic Press.