Directed evolution of novel binding proteins
First Claim
1. A method of obtaining a nucleic acid encoding a proteinaceous binding domain that binds a predetermined target material comprising:
- a) providing a variegated population of filamentous phage, each phage including a nucleic acid construct coding for a chimeric potential binding protein, each said construct comprising DNA encoding (i) a potential binding domain having one or more variable residue positions and one or more non-variable residue positions, and (ii) an outer surface transport signal for obtaining the display of the potential binding domain on the outer surface of the phage, wherein said variegated population of phage collectively display a plurality of different potential binding domains, said plurality of different potential binding domains occurring as a result of amino acid variation at said one or more variable residue positions, said phage being separable on the basis of the different potential binding domains displayed thereon;
(b) expressing said potential proteins and displaying said potential binding domains on the outer surface of said phage;
(c) contacting said phage with the predetermined target material such that said potential binding domains and the target material may interact;
(d) separating phage displaying a potential binding domain that binds the target material from phage that do not so bind, and (e) recovering at least one phage displaying on its outer surface a chimeric binding protein comprising a successful binding domain (SBD) which bound said target, said phage enclosing SBD-encoding nucleic acid, and amplifying said SBD-encoding nucleic acid in vivo or in vitro.
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Abstract
In order to obtain a novel binding protein against a chosen target, DNA molecules, each encoding a protein comprising one of a family of similar potential binding domains and a structural signal calling for the display of the protein on the outer surface of a chosen bacterial cell, bacterial spore or phage (genetic package) are introduced into a genetic package. The protein is expressed and the potential binding domain is displayed on the outer surface of the package. The cells or viruses bearing the binding domains which recognize the target molecule are isolated and amplified. The successful binding domains are then characterized. One or more of these successful binding domains is used as a model for the design of a new family of potential binding domains, and the process is repeated until a novel binding domain having a desired affinity for the target molecule is obtained. In one embodiment, the first family of potential binding domains is related to bovine pancreatic trypsin inhibitor, the genetic package is M13 phage, and the protein includes the outer surface transport signal of the M13 gene III protein.
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Citations
18 Claims
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1. A method of obtaining a nucleic acid encoding a proteinaceous binding domain that binds a predetermined target material comprising:
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a) providing a variegated population of filamentous phage, each phage including a nucleic acid construct coding for a chimeric potential binding protein, each said construct comprising DNA encoding (i) a potential binding domain having one or more variable residue positions and one or more non-variable residue positions, and (ii) an outer surface transport signal for obtaining the display of the potential binding domain on the outer surface of the phage, wherein said variegated population of phage collectively display a plurality of different potential binding domains, said plurality of different potential binding domains occurring as a result of amino acid variation at said one or more variable residue positions, said phage being separable on the basis of the different potential binding domains displayed thereon;
(b) expressing said potential proteins and displaying said potential binding domains on the outer surface of said phage;
(c) contacting said phage with the predetermined target material such that said potential binding domains and the target material may interact;
(d) separating phage displaying a potential binding domain that binds the target material from phage that do not so bind, and (e) recovering at least one phage displaying on its outer surface a chimeric binding protein comprising a successful binding domain (SBD) which bound said target, said phage enclosing SBD-encoding nucleic acid, and amplifying said SBD-encoding nucleic acid in vivo or in vitro. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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Specification