Development of polyol-responsive antibody mimetics for single-step protein purification

- Novel, polyol-responsive (elutable) antibody mimetics, nanoCLAMPs, are described.
- nanoCLAMPs were produced and incorporated into affinity chromatography resins.
- Targets were purified in a single chromatography step from spiked crude lysates.
- Mild polyol elution buffer preserved structure and activity of eluted proteins.
- nanoCLAMPs are an ideal starting point for structural and functional studies.

The purification of functional proteins is a critical pre-requisite for many experimental assays. Immunoaffinity chromatography, one of the fastest and most efficient purification procedures available, is often limited by elution conditions that disrupt structure and destroy enzymatic activity. To address this limitation, we developed polyol-responsive antibody mimetics, termed nanoCLAMPs, based on a 16 kDa carbohydrate binding module domain from Clostridium perfringens hyaluronidase. nanoCLAMPs bind targets with nanomolar affinity and high selectivity yet release their targets when exposed to a neutral polyol-containing buffer, a composition others have shown to preserve quaternary structure and enzymatic activity. We screened a phage display library for nanoCLAMPs recognizing several target proteins, produced affinity resins with the resulting nanoCLAMPs, and successfully purified functional target proteins by single-step affinity chromatography and polyol elution. To our knowledge, nanoCLAMPs constitute the first antibody mimetics demonstrated to be polyol-responsive.