Avidity-mediated virus separation using a hyperthermophilic affinity ligand

Immunoaffinity separation of large multivalent species such as viruses is limited by the stringent elution conditions necessary to overcome their strong and highly avid interaction with immobilized affinity ligands on the capture surface. Here we present an alternate strategy that harnesses the avidity effect to overcome this limitation. Red clover necrotic mosaic virus (RCNMV), a plant virus relevant to drug delivery applications, was chosen as a model target for this study. An RCNMV binding protein (RBP) with modest binding affinity (K(D) ~100 nM) was generated through mutagenesis of the Sso7d protein from Sulfolobus solfataricus and used as the affinity ligand. In our separation scheme, RCNMV is captured by a highly avid interaction with RBP immobilized on a nickel surface through a hexahistidine (6xHis) tag. Subsequently, disruption of the multivalent interaction and release of RCNMV is achieved by elution of RBP from the nickel surface. Finally, RCNMV is separated from RBP by exploiting the large difference in their molecular weights (~8 MDa vs. ~10 kDa). Our strategy not only eliminates the need for harsh elution conditions, but also bypasses chemical conjugation of the affinity ligand to the capture surface. Stable non-antibody affinity ligands to a wide spectrum of targets can be generated through mutagenesis of Sso7d and other hyperthermophilic proteins. Therefore, our approach may be broadly relevant to cases where capture of large multivalent species from complex mixtures and subsequent release without the use of harsh elution conditions is necessary.