Method involving display of protein binding pairs on the surface of bacterial pili and bacteriophage
AssigneeDade International Inc.
InventorsGrace P. Huang, Peter R. Rhode, Jeffrey R. Stinson, Hing C. Wong
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Abstract
The present invention relates to a fusion protein, comprising a pilin protein or a portion thereof and a heterologous polypeptide (target protein). In a preferred embodiment it relates to a method for displaying the target protein on the outer surface of a bacterial host cell capable of forming pilus. In certain embodiments, it is desirable that the pilus is a receptor for bacteriophage attachment and infection. The F pilus is preferred.
Claims14
A method for isolating a member of a specific binding pair, comprising said member and an other member, said method comprising:
- (a) expressing a fusion protein in a bacterial host which forms a pilus that is a receptor for bacteriophage attachment and infection, said fusion protein encoded by a chimeric DNA comprising a DNA segment encoding a leader amino acid sequence which mediates secretion of the fusion protein, a DNA segment encoding a pilin subunit which forms a pilus, and a DNA segment encoding said member;
- (b) contacting the bacterial host of step (a) with a bacteriophage displaying an attachment protein wherein DNA encoding a pilin interaction domain has been substituted with DNA encoding the other member of the specific binding pair;
- and
- (c) selecting and then isolating bacterial host cells recognized by the bacteriophage, thereby isolating said member of the specific binding pair.
A method of screening for compounds affecting specific binding pair interaction comprising:
- (a) contacting a bacterial cell having displayed on its surface a fusion protein comprised of pilin and a member of a specific binding pair, said specific binding pair comprised of the member and an other member, with a bacteriophage altered such that a pilin interaction domain is substituted by the other member of the specific binding pair;
- (b) adding a test compound;
- and
- (c) measuring an effect of the test compound on the interaction between the bacteriophage and the bacterial cell, and thereby determining whether the test compound affects the specific binding pair interaction.
A method of screening for a member of a specific binding pair having increased binding affinity for an other member of the specific binding pair comprising:
- (a) contacting a display bacterium displaying a fusion protein comprised of pilin and the other member of the specific binding pair with a bacteriophage altered such that a normal pilin binding domain is substituted with a protein from a library of proteins containing the member of the specific binding pair under conditions that allow bacteriophage infection;
- and
- (b) identifying specific binding pairs having increased binding affinity by identifying bacteriophage having an increased frequency of infectivity.
Dependencies are inferred from the claim text.