We have generated a stable (Tm ~ 83 deg.C) binding protein for Red clover necrotic mosaic virus (RCNMV), a plant virus, through mutagenesis of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus. The Sso7d-based RCNMV-binding protein (RBP) was then used to purify RCNMV from plant sap, using a variation of affinity chromatography. In this scheme � termed avidity chromatography � RCNMV from plant sap is captured on a nickel column that is pre-loaded with hexahistidine tagged RBP. The highly avid interaction between RCNMV and immobilized RBP ensures efficient capture of RCNMV despite modest binding affinity (KD ~ 100 nM) of the RBP-RCNMV interaction. Subsequently, RBP is eluted from the column along with RCNMV; a single density gradient ultracentrifugation step is used to separate the RBP from RCNMV, as well as concentrate pure RCNMV. This purification scheme precludes the need for harsh elution conditions that are typically required for affinity chromatography of viruses and also eliminates the need for chemical conjugation of the affinity ligand to a resin. Thus, Sso7d-based binding proteins are well-suited for use as affinity ligands for viruses in general, and may reduce the cost and complexity of virus purification.

Hyperthermophilic affinity ligand for virus purification by avidity chromatography

243rd National Meeting (2012)

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