US 5,837,500 A · Directed evolution of novel binding proteins

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.

Claims43

Dependent claims

A method of obtaining a nuclecic acid encoding a proteinaceous binding domain that binds a predetermined target material comprising:

a) preparing 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 which is a mutant of a predetermined parental binding domain, and (ii) an outer surface transport signs 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, the differentiation among said plurality of different potential binding domains occuring through the at least partially random variation of one or more predetermined amino acid position of said parental binding domain to randomly obtain at each said position an amino belonging to a predetermined set of two or more amino acids, the amino acids of said set occuring at said position in predetermined expected proportions, said phage being separable on the basis of the potential binding domains displayed thereon;
b) causing the expression of said potential proteins and the display of 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 interact;
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.

2 more elements

A method of separating a predetermined target material from a predetermined contaminating material which comprises

(I) obtaining a nucleic acid encoding a binding protein having a proteinaceous binding domain that binds to said predetermined target material substantially more strongly than to said predetermined contaminating material, by a method comprising:
a) preparing a variegated population of amplifiable genetic packages, said genetic packages being selected from the group consisting of cells, spores and viruses, each said genetic package being genetically alterable and having an outer surface including a genetically determined outer surface protein, each package including a first nucleic acid construct coding for a chimeric potential binding protein, each said chimeric protein comprising and each said construct comprising DNA encoding (i) a potential binding domain which is a mutant of a predetermined domain of a predetermined parental protein other than a single chain antibody, and (ii) an outer surface transport signal for obtaining the display of the potential binding domain on the outer surface of the genetic package, the expression of which construct results in the display of said chimeric potential binding protein and its potential binding domain on the outer surface of said genetic package:
and wherein said variegated population of genetic packages collectively display a plurality of different potential binding domains, the differentiation among said plurality of different potential binding domains occuring through the at least partially random variation of one or more predetermined amino acid positions of said parental binding domain to randomly obtain at each said position an amino acid belonging to a predetermined set of two or more amino acids, the amino acids of said set occuring at said position in statistically predetermined expected proportions, said genetic packages being amplifiable in cell culture and separable on the basis of the potential binding domain displayed thereon,
(b) causing the expression of said chimeric potential binding proteins and the display of said potential binding domains on the outer surface of said packages;
(c) selecting for binding to the predetermined target material by
(i) contacting said package with the predetermined target material such that said potential binding domains and the target material may interact;
and
(ii) separating packages displaying a potential binding domain that binds the target material from packages that do not so bind, and
(d) selecting for non-binding to the predetermined contaminating material by
(i) contacting said package with the predetermined contaminating material such that said potential binding domains and the contaminating material may interact;
(ii) separating packages dispalying a potential binding domain that binds the contaminating material from packages that do not so bind, and
(e) recovering at least one package displaying on its outer surface a chimeric binding protein comprising a successful binding domain (SBD) which bound said target material in step (c) and less strongly bound, if at all, said contaminating material in step (d), said package comprising nucleic acid encoding said successful binding domain, and amplifying said SBD-encoding nucleic acid in vivo or in vitro;
(II) producing a binding protein which is either said chimeric binding protein or a second binding protein having essentially the same binding domain;
(III) immobilizing or labeling said binding protein of (II), so as to form an affinity separation reagent;
and
(IV) contacting a mixture comprising said target material and said contaminating material with said reagent such that said target material and said reagent can interact, and separating said target material from said contaminating material on the basis of their affinity for said affinity separation reagent.

13 more elements

A method of separating a predetermined target material from a predetermined contaminating material which comprises

(I) obtaining a nucleic acid encoding a binding protein having a proteinaceous binding domain that binds to said predetermined target material substantially more strongly than to said predetermined contaminating material, by a method comprising:
a) preparing a variegated population of phage, each phage including a first nucleic acid construct coding for a chimeric potential binding protein, each said chimeric protein comprising and each said construct comprising DNA encoding
(i) a potential binding domain which is a mutant of a predetermined domain of a predetermined parental protein, and (ii) an outer surface transport signal for obtaining the display of the potential binding domain on the outer surface of the phage, the expression of which construct results in the display of said chimeric potential binding protein and its potential binding domain on the outer surface of said phage:
and wherein said variegated population of phage collectively display a plurality of different potential binding domains, the differentiation among said plurality of different potential binding domains occuring through the at least partially random variation of one or more predetermined amino acid position of said parental binding domain to randomly obtain at each said position an amino acid belonging to a predetermined set of two or more amino acids, the amino acids of said set occuring at said position in statistically predetermined expected proportions, said phage being amplifiable by cultivation of phage-infected cells in cell culture and separable on the beats of the potential binding domain displayed thereon,
(b) causing the expression of said chimeric potential binding proteins and the display of said potential binding domains on the outer surface of said phage;
(c) selecting for binding to the predetermined target material by
(i) contacting said phage with the predetermined target material such that said potential binding domains and the target material may interact;
and
(ii) separating packages displaying a potential binding domain that binds the target material from packages that do not so bind, and
(d) selecting for non-binding to the predetermined contaminating material by
(i) contacting said phage with the predetermined contaminating material such that said potential binding domains and the contaminating material may interact;
(ii) separating packages dispalying a potential binding domain that binds the contaminating 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 material in step (c) and less strongly bound said contaminating material in step (d), said phage comprising nucleic acid encoding said successful binding domain, and amplifying said SBD-encoding nucleic acid in vivo or in vitro;
(II) producing a binding protein which is either said chimeric binding protein or a second binding protein having essentially the same binding domain;
(III) immobilizing or labeling said binding protein of (II), so as to form an affinity separation reagent;
and
(IV) contacting a mixture comprising said target material and said contaminating material with said reagent such that said target material and said reagent can interact, and separating said target material from said contaminating material on the basis of their affinity for said affinity separation reagent.

14 more elements

A mehtod of obtaining a nucleic acid encoding a binding protein haing a proteinaceous binding domain that binds a predetermined target material comprising:

a) preparing a variegated population of amplifiable genetic packages, said genetic packages being selected from the group consisting of cells, spores and viruses, each said genetic package being gentically alterable and having an outer surface including a genetically determined outer surface protein, each package including a first nucleic acid construct coding for a chimeric potential binding protein, each said chimeric protein comprising and each said construct comprising nucleic acid encoding (i) a potential binding domain which is a mutant of a predetermined domain of a predetermined parental protein other than a sigle chain antibdy, and (ii) an outer surface transport signal for obtaining the display of the potential binding domain on the outer surface of the genetic package, the expression of which construct results in the display of said chimeric potential binding protein and its potential binding domain on the outer surface of said genetic package;
and wherein said varigated population of genetic packages collectively display a plurality of different potential binding domains, the differentiation among said plurality of different potential binding domains occuring through the at least partially random variation of one or more predetermined amino acid positions of said parental binding domain to randomly obtain at each said position an amino acid belonging to a predetermined set two or more amino acids, the amino acids of acid of said set occuring at said position in statistically predetermined expected proportions, said genetic packages being separable on the basis of the potential binding domain displayed thereon;
b) causing the expression of said chimeric potential binding proteins and the display of said potential binding domains on the outer surface of said packages;
c) contacting said package with predetermined target material such that said potential binding domains and the target material may interact;
d) separating packages displaying a potential binding domain that binds the target material from packages that do not so bind, and
e) recovering at least one package displaying on its outer surface a chimeric binding protein comprising a successful binding domain (SBD) which bound said target, said package comprising nucleic acid encoding said successful binding domain, and amplifying said SBD-encoding nucleic acid in vivo or in vitro.

3 more elements

Dependencies are inferred from the claim text.

Directed evolution of novel binding proteins

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Directed evolution of novel binding proteins

Life of this patent

Abstract

Claims43