Antibodies, the quintessential biological recognition molecules, are not ideal for many applications because of their large size, complex modifications, and thermal and chemical instability. Identifying alternative scaffolds that can be evolved into tight specific binding molecules with improved physical properties is of increasing interest for applications in diagnostics and as therapeutics. Hyperthermophilic organisms, such as Sulfolobus solfataricus, are an attractive source of highly stable proteins as starting points for alternative antibody scaffolds. We describe the first phage display libraries utilizing the Sulfolobus solfataricus protein Sso7d as an alternative antibody scaffold. Ss07d is a small (~7KDa, 63 amino acids), cysteine free DNA-binding protein with a melting temperature of nearly 100˚C. This protein has been employed in yeast display directed evolution experiments to isolate tight binding proteins against diverse targets. We constructed a 1010 member Ss07d mutant library by Kunkel mutagenesis. Multiple tight binding Ss07d variants have been selected from this library against a diverse set of protein targets, including bovine serum albumin, rabbit serum albumin, neutravidin, green fluorescent protein, and C - reactive protein. Equilibrium dissociation constants (KD) in the nanomolar to low micromolar range were measured via competitive ELISA and ITC. The selected Ss07d variants displayed on p3 of M13 phage maintain their binding affinity after being heated to 70˚C. Phages decorated with hyperthermostable binding proteins have potential applications as sensors in resource limited areas.

Promising thermostable alternative scaffold protein for M13 phage display

249th National Meeting (2015)

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