Phagemids coexpressing a surface receptor and a surface heterologous protein
First Claim
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1. A filamentous phage comprising:
- a) a heterologous polypeptide fused to a first filamentous phage coat protein membrane anchor; and
b) a heterodimeric receptor comprised of first and second receptor polypeptides wherein one of said receptor polypeptides is fused to a second filamentous phage coat protein membrane anchor,wherein said first and second coat protein membrane anchors are selected from the group consisting of cpllI and cpVIII.
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Abstract
A filamentous phage is described comprising a matrix that includes a heterologous polypeptide fused to a first filamentous phage coat protein membrane anchor and a heterodimeric receptor comprised of first and second receptor polypeptides, wherein one of the receptor polypeptides is fused to a second filamentous phage coat protein membrane anchor. Filamentous phage expressing anchored heterodimeric receptors and dimers of heterologous polypeptides where a first subunit of the dimer is fused to a coat protein membrane anchor and the second subunit of the dimer is soluble heteromeric receptor are also described.
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Citations
45 Claims
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1. A filamentous phage comprising:
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a) a heterologous polypeptide fused to a first filamentous phage coat protein membrane anchor; and b) a heterodimeric receptor comprised of first and second receptor polypeptides wherein one of said receptor polypeptides is fused to a second filamentous phage coat protein membrane anchor, wherein said first and second coat protein membrane anchors are selected from the group consisting of cpllI and cpVIII. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A library of filamentous phage particles wherein each filamentous phage particle comprises:
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a) a heterologous polypeptide fused to a first filamentous phage coat protein membrane anchor; and b) a heterodimeric receptor comprised of first and second receptor polypeptides wherein one of said receptor polypeptides is fused to a second filamentous phage coat protein membrane anchor, wherein said first and second coat protein membrane anchors are selected from the group consisting of cpIII and cpVIII. - View Dependent Claims (11, 12, 13, 14, 15)
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16. A method for producing a filamentous phage particle having on the particle surface (i) a first fusion polypeptide and (ii) a heterodimeric receptor consisting of first and second receptor polypeptides, which method comprises the steps of:
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a) introducing into a prokaryotic host cell permissive for filamentous phage replication a first vector comprising a nucleotide sequence which expresses said first fusion polypeptide, said first fusion polypeptide comprising a heterologous polypeptide fused to a first filamentous phage coat protein membrane anchor; b) introducing into said prokaryotic host cell a second vector for expressing said heterodimeric receptor comprising a nucleotide sequence which expresses said first and second receptor polypeptides, wherein one of said receptor polypeptides is fused to a second filamentous phage coat protein membrane anchor; and c) maintaining said prokaryotic host cell containing said introduced first and second vectors under conditions sufficient for filamentous phage production and under conditions sufficient for expression of said first fusion polypeptide and expression of said heterodimeric receptor, thereby forming said phage particle, wherein said first and second coat protein membrane anchors are selected from the group consisting of cpIII and cpVIII. - View Dependent Claims (17, 18, 19, 20, 21, 22, 23, 24, 25)
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26. A method for producing a filamentous phage particle containing a surface-exposed heterodimeric receptor having a preselected binding specificity, which method comprises the steps of:
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a) providing a library of filamentous phage particles wherein each filamentous phage particle comprises; i) an indicator polypeptide fused to a first filamentous phage coat protein membrane anchor; and ii) a heterodimeric receptor comprised of first and second receptor polypeptides wherein one of said receptor polypeptides is fused to a second filamentous phage coat protein membrane anchor, said heterodimeric receptor able to bind to a preselected ligand; b) adsorbing members of said provided library onto a plurality of preselected ligand molecules in the solid phase to form a plurality of solid-phase adsorbed phage particles; c) assaying said solid phase for the presence of said indicator polypeptide, and thereby the presence of a solid-phase adsorbed phage particle containing said surface-exposed heterodimeric receptor having a preselected binding specificity; and d) recovering said solid-phase adsorbed phage particle that contains said heterodimeric receptor, wherein said first and second coat protein membrane anchors are selected from the group consisting of cpIII and cpVIII. - View Dependent Claims (27, 28, 29, 30, 31, 32, 33, 34)
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35. A method for detecting the presence of a preselected ligand in a sample comprising the steps of:
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a) contacting said sample with a filamentous phage particle comprising; i) an indicator polypeptide fused to a first filamentous phage coat protein membrane anchor, and ii) a heterodimeric receptor comprised of first and second receptor polypeptides wherein one of said receptor polypeptides is fused to a second filamentous phage coat protein membrane anchor, said heterodimeric receptor able to bind to said preselected ligand;
to form a ligand binding reaction admixture;b) maintaining said admixture under conditions sufficient for said heterodimeric receptor to bind to said ligand and form a ligand-filamentous phage particle complex; and c) detecting the presence of said indicator polypeptide on said complex, thereby detecting said preselected ligand, wherein said first and second coat protein membrane anchors are selected from the group consisting of cpIII and cpVIII. - View Dependent Claims (36, 37, 38, 39, 40, 41, 42)
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43. A vector for expressing first and second polypeptide subunits of an alkaline phosphatase dimer which dimer upon expression assembles on the surface of a filamentous phage particle, said vector comprising a nucleotide sequence that encodes:
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a) a suppressor tRNA gene which expresses a suppressor tRNA molecule; and b) an expression cassette for expressing said first and second polypeptide subunits, said expression cassette comprising; i) a transcriptional promoter and transcriptional terminator for producing a messenger RNA transcript that encodes said first and second polypeptide subunits; ii) a first open reading frame that encodes soluble alkaline phosphatase beginning with a translational initiator and ending with a nonsense chain termination codon selected from the group consisting of amber, ochre and opal; and iii) a second open reading frame operatively linked downstream to said first open reading frame, said second open reading frame encoding a filamentous phage coat protein membrane anchor such that upon suppression of the nonsense chain termination codon by said suppressor tRNA molecule, said first and second open reading frames are translated as one polypeptide, the translated polypeptide being a fusion protein having alkaline phosphatase operatively linked in frame with the filamentous phage coat protein membrane anchor, wherein said coat protein membrane anchor is selected from the group consisting of cpIII and cpVIII. - View Dependent Claims (44, 45)
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Specification