title: Phage display selection of tight specific binding variants from a hyperthermostable Sso7d scaffold protein library.
abstract: 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 may be evolved into tight, specific binding molecules with improved physical properties is of increasing interest, particularly for biomedical applications in resource-limited environments. Hyperthermophilic organisms, such as Sulfolobus solfataricus, are an attractive source of highly stable proteins that may serve as starting points for alternative molecular recognition scaffolds. We describe the first application of phage display to identify binding proteins based on the S. solfataricus protein Sso7d scaffold. Sso7d is a small cysteine-free DNA-binding protein (approximately 7 kDa, 63 amino acids), with a melting temperature of nearly 100 °C. Tight-binding Sso7d variants were selected for a diverse set of protein targets from a 10(10) member library, demonstrating the versatility of the scaffold. These Sso7d variants are able to discriminate among closely related human, bovine and rabbit serum albumins. Equilibrium dissociation constants in the nanomolar to low micromolar range were measured via competitive ELISA. Importantly, the Sso7d variants continue to bind their targets in the absence of the phage context. Furthermore, phage-displayed Sso7d variants retain their binding affinity after exposure to temperatures up to 70 °C. Taken together, our results suggest that the Sso7d scaffold will be a complementary addition to the range of non-antibody scaffold proteins that may be utilized in phage display. Variants of hyperthermostable binding proteins have potential applications in diagnostics and therapeutics for environments with extreme conditions of storage and deployment.
authors.affiliation: Department of Chemical and Biological Engineering, Colorado State University, Fort Collins, CO, USA.
authors.forename: Ning
authors.initials: N
authors.lastname: Zhao
authors.affiliation: Department of Chemical and Biological Engineering, Colorado State University, Fort Collins, CO, USA.
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Schmitt
authors.affiliation: Department of Chemical and Biological Engineering, Colorado State University, Fort Collins, CO, USA.|Department of Chemistry, Colorado State University, Fort Collins, CO, USA.|School of Biomedical Engineering, Colorado State University, Fort Collins, CO, USA.
authors.forename: John D
authors.identifier: http://orcid.org/0000-0001-9809-6140
authors.initials: JD
authors.lastname: Fisk
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D002151:Calorimetry / Q000379:methods; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D061505:Cell Surface Display Techniques / Q000379:methods*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D019151:Peptide Library; D011485:Protein Binding; D012709:Serum Albumin / Q000235:genetics / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000235:genetics / Q000378:metabolism; D048229:Sulfolobus solfataricus / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
chemical_list: D019843:Archaeal Proteins; D004268:DNA-Binding Proteins; D019151:Peptide Library; D012709:Serum Albumin; C093228:Sso7d protein, Sulfolobus; D049452:Green Fluorescent Proteins; D012710:Serum Albumin, Bovine
keywords: alternative scaffold; combinatorial libraries; hyperthermophilic proteins; molecular recognition; phage display