Display of disulfide linked dimeric proteins in filamentous phage
First Claim
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1. A replicable phage vector for the display of a functional, multimeric, interchain disulfide linked protein on the surface of a filamentous phage particle;
- comprising nucleic acid sequences encoding an inducible promoter and nucleic acid sequences encoding a first polypeptide chain and a second polypeptide chain wherein each chain consists of amino acid residues according to the following formula;
N-(a bacterial secretion signal)-(Formula I)-(a phage coat protein)-Cwherein N is the N-terminus and C is the C-terminus;
wherein the phage coat protein is a phage pIX or pVII protein;
wherein Formula I is V1o-Pepa-Flexn-V2m-Hinge-CH2—
CH3;
wherein Pep represents a bioactive peptide or polypeptide capable of specifically recognizing a target;
wherein Flex is an optional flexible linker polypeptide that provides structural flexibility;
wherein V1 and V2 are bracketing sequences;
wherein Hinge is at least a portion of an immunoglobulin hinge region selected from the group consisting of;
SEQ ID NO;
1-4, SEQ ID NO 1 amino acid residues 11-15, SEQ ID NO 2 amino acid residues 8-12, SEQ ID NO 3 amino acid residues 13-61 and SEQ ID NO 4 amino acid residues 8-12;
wherein CH2 is at least a portion of an immunoglobulin CH2 constant region;
wherein CH3 is at least a portion of an immunoglobulin CH3 constant region;
wherein m, n and o can be zero or can be an integer between 1 and 10, and a can be an integer from 1 to 10;
wherein said encoded Hinge amino acid sequence comprises at least one cysteine residue;
wherein a cysteine residue on the first polypeptide chain is capable of becoming oxidatively bonded to a Hinge cysteine residue on the second polypeptide chain; and
whereby the cysteine bond so formed is an interchain disulfide of the functional multimeric structure being displayed on the surface of the same filamentous phage particle.
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Abstract
Methods are provided for the display of a complex homodimer protein on the surface of a bacteriophage particle and combinatorial synthetic libraries of such proteins displayed as a fusion polypeptide with filamentous phage pIX coat protein. Heterodimeric or more complex interchain bonded structure, such as disulfide-linked, multimeric proteins, may be displayed using the method of the invention.
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9 Claims
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1. A replicable phage vector for the display of a functional, multimeric, interchain disulfide linked protein on the surface of a filamentous phage particle;
- comprising nucleic acid sequences encoding an inducible promoter and nucleic acid sequences encoding a first polypeptide chain and a second polypeptide chain wherein each chain consists of amino acid residues according to the following formula;
N-(a bacterial secretion signal)-(Formula I)-(a phage coat protein)-Cwherein N is the N-terminus and C is the C-terminus; wherein the phage coat protein is a phage pIX or pVII protein; wherein Formula I is V1o-Pepa-Flexn-V2m-Hinge-CH2—
CH3;wherein Pep represents a bioactive peptide or polypeptide capable of specifically recognizing a target; wherein Flex is an optional flexible linker polypeptide that provides structural flexibility; wherein V1 and V2 are bracketing sequences; wherein Hinge is at least a portion of an immunoglobulin hinge region selected from the group consisting of;
SEQ ID NO;
1-4, SEQ ID NO 1 amino acid residues 11-15, SEQ ID NO 2 amino acid residues 8-12, SEQ ID NO 3 amino acid residues 13-61 and SEQ ID NO 4 amino acid residues 8-12;wherein CH2 is at least a portion of an immunoglobulin CH2 constant region; wherein CH3 is at least a portion of an immunoglobulin CH3 constant region; wherein m, n and o can be zero or can be an integer between 1 and 10, and a can be an integer from 1 to 10; wherein said encoded Hinge amino acid sequence comprises at least one cysteine residue; wherein a cysteine residue on the first polypeptide chain is capable of becoming oxidatively bonded to a Hinge cysteine residue on the second polypeptide chain; and whereby the cysteine bond so formed is an interchain disulfide of the functional multimeric structure being displayed on the surface of the same filamentous phage particle. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
- comprising nucleic acid sequences encoding an inducible promoter and nucleic acid sequences encoding a first polypeptide chain and a second polypeptide chain wherein each chain consists of amino acid residues according to the following formula;
Specification