title: Targeted Mutagenesis and Combinatorial Library Screening Enables Control of Protein Orientation on Surfaces and Increased Activity of Adsorbed Proteins.
abstract: While nonspecific adsorption is widely used for immobilizing proteins on solid surfaces, the random nature of protein adsorption may reduce the activity of immobilized proteins due to occlusion of the active site. We hypothesized that the orientation a protein assumes on a given surface can be controlled by systematically introducing mutations into a region distant from its active site, thereby retaining activity of the immobilized protein. To test this hypothesis, we generated a combinatorial protein library by randomizing six targeted residues in a binding protein derived from highly stable, nonimmunoglobulin Sso7d scaffold; mutations were targeted in a region that is distant from the binding site. This library was screened to isolate binders that retain binding to its cognate target (chicken immunoglobulin Y, cIgY) as well as exhibit adsorption on unmodified silica at pH 7.4 and high ionic strength conditions. A single mutant, Sso7d-2B5, was selected for further characterization. Sso7d-2B5 retained binding to cIgY with an apparent dissociation constant similar to that of the parent protein; both mutant and parent proteins saturated the surface of silica with similar densities. Strikingly, however, silica beads coated with Sso7d-2B5 could achieve up to 7-fold higher capture of cIgY than beads coated with the parent protein. These results strongly suggest that mutations introduced in Sso7d-2B5 alter its orientation relative to the parent protein, when adsorbed on silica surfaces. Our approach also provides a generalizable strategy for introducing mutations in proteins so as to improve their activity upon immobilization, and has direct relevance to development of protein-based biosensors and biocatalysts.
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University , Raleigh, North Carolina 27695, United States.
authors.forename: Carlos A
authors.initials: CA
authors.lastname: Cruz-Teran
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University , Raleigh, North Carolina 27695, United States.
authors.forename: Kevin B
authors.initials: KB
authors.lastname: Carlin
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University , Raleigh, North Carolina 27695, United States.
authors.forename: Kirill
authors.initials: K
authors.lastname: Efimenko
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University , Raleigh, North Carolina 27695, United States.
authors.forename: Jan
authors.initials: J
authors.lastname: Genzer
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University , Raleigh, North Carolina 27695, United States.
authors.forename: Balaji M
authors.initials: BM
authors.lastname: Rao
mesh_terms: D000327:Adsorption; D000818:Animals; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002645:Chickens; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055767:Immobilized Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007136:Immunoglobulins / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019151:Peptide Library; D011485:Protein Binding; D012822:Silicon Dioxide; D013499:Surface Properties
chemical_list: D019843:Archaeal Proteins; D004268:DNA-Binding Proteins; C016512:IgY; D055767:Immobilized Proteins; D007136:Immunoglobulins; D050505:Mutant Proteins; D019151:Peptide Library; C093228:Sso7d protein, Sulfolobus; D012822:Silicon Dioxide