title: Highly stable binding proteins derived from the hyperthermophilic Sso7d scaffold.
abstract: We have shown that highly stable binding proteins for a wide spectrum of targets can be generated through mutagenesis of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus. Sso7d is a small (~7 kDa, 63 amino acids) DNA-binding protein that lacks cysteine residues and has a melting temperature of nearly 100 °C. We generated a library of 10(8) Sso7d mutants by randomizing 10 amino acid residues on the DNA-binding surface of Sso7d, using yeast surface display. Binding proteins for a diverse set of model targets could be isolated from this library; our chosen targets included a small organic molecule (fluorescein), a 12 amino acid peptide fragment from the C-terminus of β-catenin, the model proteins hen egg lysozyme and streptavidin, and immunoglobulins from chicken and mouse. Without the application of any affinity maturation strategy, the binding proteins isolated had equilibrium dissociation constants in the nanomolar to micromolar range. Further, Sso7d-derived binding proteins could discriminate between closely related immunoglobulins. Mutant proteins based on Sso7d were expressed at high yields in the Escherichia coli cytoplasm. Despite extensive mutagenesis, Sso7d mutants have high thermal stability; five of six mutants analyzed have melting temperatures >89 °C. They are also resistant to chemical denaturation by guanidine hydrochloride and retain their secondary structure after extended incubation at extreme pH values. Because of their favorable properties, such as ease of recombinant expression, and high thermal, chemical and pH stability, Sso7d-derived binding proteins will have wide applicability in several areas of biotechnology and medicine.
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Nimish
authors.initials: N
authors.lastname: Gera
authors.forename: Mahmud
authors.initials: M
authors.lastname: Hussain
authors.forename: Robert C
authors.initials: RC
authors.lastname: Wright
authors.forename: Balaji M
authors.initials: BM
authors.lastname: Rao
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002645:Chickens; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019793:Fluorescein / Q000737:chemistry / Q000378:metabolism; D007136:Immunoglobulins / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism; D048229:Sulfolobus solfataricus / Q000737:chemistry*; D051176:beta Catenin / Q000737:chemistry / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D004268:DNA-Binding Proteins; D007136:Immunoglobulins; C093228:Sso7d protein, Sulfolobus; D051176:beta Catenin; D019809:Streptavidin; C421864:hen egg lysozyme; D009113:Muramidase; D019793:Fluorescein