title: Selecting proteins with improved stability by a phage-based method.
abstract: We describe a method for the stabilization of proteins that links the protease resistance of stabilized variants of a protein with the infectivity of a filamentous phage. A repertoire of variants of the protein to be stabilized is inserted between two domains (N2 and CT) of the gene-3-protein of the fd phage. The infectivity of fd phage is lost when the three domains are disconnected by the proteolytic cleavage of unstable protein inserts. Rounds of in vitro proteolysis, infection, and propagation can thus be performed to enrich those phage containing the most stable variants of the protein insert. This strategy discriminates between variants of a model protein (ribonuclease T1) differing in conformational stability and selects from a large repertoire variants that are only marginally more stable than others. Because fd phage are exceptionally stable and the proteolysis in the selection step takes place in vitro a wide range of solvent conditions can be used, tailored for the protein to be stabilized.
authors.affiliation: Biochemisches Laboratorium, Universität Bayreuth, Germany.
authors.forename: V
authors.initials: V
authors.lastname: Sieber
authors.forename: A
authors.initials: A
authors.lastname: Plückthun
authors.forename: F X
authors.initials: FX
authors.lastname: Schmid
mesh_terms: D001483:Base Sequence; D036022:Capsid Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics; D006868:Hydrolysis; D017912:Inovirus / Q000235:genetics* / Q000472:pathogenicity; D016297:Mutagenesis, Site-Directed; D006163:Ribonuclease T1 / Q000235:genetics* / Q000378:metabolism; D013816:Thermodynamics; D014760:Viral Fusion Proteins / Q000235:genetics
chemical_list: D036022:Capsid Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D014760:Viral Fusion Proteins; D006163:Ribonuclease T1