Method of selection of proteolytic cleavage sites by directed evolution and phagemid display
First Claim
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1. A method for selecting novel polypeptides comprising:
- (a) constructing a replicable expression vector comprising a transcription regulatory element operably linked to a gene fusion, wherein the gene fusion comprises;
(i) a first gene encoding a polypeptide;
(ii) a second gene encoding a substrate peptide; and
(iii) a third gene encoding at least a portion of a phage coat protein,wherein the 3'"'"' end of the first gene is linked to the 5'"'"' end of the second gene, and the 3'"'"' end of the second gene is linked to the 5'"'"' end of the third gene;
(b) mutating the vector at one or more selected positions within the second gene thereby forming a family of related plasmids encoding substrate peptides;
(c) transforming suitable host cells with the plasmids;
(d) infecting the transformed host cells with a helper phage having a gene encoding the phage coat protein;
(e) culturing the transformed infected host cells under conditions suitable for forming recombinant phagemid particles containing at least a portion of the plasmid and capable of transforming the host, the conditions adjusted so that no more than a minor amount of phagemid particles display more than one copy of the fusion protein on the surface of the particle;
(f) exposing the phagemid particles to at least one protease to provide a family of protease treated phagemid particles;
(g) contacting the family of protease treated phagemid particles with an affinity molecule, wherein the affinity molecule has affinity for the polypeptide encoded by the first gene; and
(h) separating the phagemid particles that bind to the affinity molecule from those that do not.
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Abstract
A method for identifying and selecting novel substrates for enzymes is provided. The method comprises constructing a gene fusion comprising DNA encoding a polypeptide fused to DNA encoding a substrate peptide, which in turn is fused to DNA encoding at least a portion of a phage coat protein. The DNA encoding the substrate peptide is mutated at one or more codons thereby generating a family of mutants. The fusion protein is expressed on the surface of a phagemid particle and subjected to chemical or enzymatic modification of the substrate peptide. Those phagemid particles which have been modified are then separated from those that have not.
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Citations
25 Claims
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1. A method for selecting novel polypeptides comprising:
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(a) constructing a replicable expression vector comprising a transcription regulatory element operably linked to a gene fusion, wherein the gene fusion comprises; (i) a first gene encoding a polypeptide; (ii) a second gene encoding a substrate peptide; and (iii) a third gene encoding at least a portion of a phage coat protein, wherein the 3'"'"' end of the first gene is linked to the 5'"'"' end of the second gene, and the 3'"'"' end of the second gene is linked to the 5'"'"' end of the third gene; (b) mutating the vector at one or more selected positions within the second gene thereby forming a family of related plasmids encoding substrate peptides; (c) transforming suitable host cells with the plasmids; (d) infecting the transformed host cells with a helper phage having a gene encoding the phage coat protein; (e) culturing the transformed infected host cells under conditions suitable for forming recombinant phagemid particles containing at least a portion of the plasmid and capable of transforming the host, the conditions adjusted so that no more than a minor amount of phagemid particles display more than one copy of the fusion protein on the surface of the particle; (f) exposing the phagemid particles to at least one protease to provide a family of protease treated phagemid particles; (g) contacting the family of protease treated phagemid particles with an affinity molecule, wherein the affinity molecule has affinity for the polypeptide encoded by the first gene; and (h) separating the phagemid particles that bind to the affinity molecule from those that do not. - View Dependent Claims (2, 3, 4, 5, 6, 8, 9, 10, 13, 14, 15, 16, 17, 18)
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7. The method of claim I wherein the first gene encodes a polypeptide selected from the group consisting of;
- human growth hormone (hGH), N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin A-chain, insulin B-chain, proinsulin, relaxin A-chain, relaxin B-chain, prorelaxin, a glycoprotein hormone follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), leutinizing hormone (LH), glycoprotein hormone receptors, calcitonin, glucagon, factor VIII, an antibody, lung surfactant, urokinase, streptokinase, thrombin, hemopoietic growth factor, tumor necrosis factor-alpha, tumor necrosis factor-beta, enkephalinase, human serum albumin, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, a microbial protein, betalactamase, tissue factor protein, inhibin, activin, vascular endothelial growth factor, hormone receptors, growth factor receptors integrin, thrombopoietin, protein A. protein D, rheumatoid factors, nerve growth factors, NGF-b, platelet-growth factor, transforming growth factors (TGF), TGF-alpha, TGF-beta, insulin-like growth factor-I, insulin-like growth factor-II, insulin-like growth factor binding proteins, CD-4, DNase, latency associated peptide, erythropoietin, osteoinductive factors, interferons, interferon-alpha, interferon-beta, interferon-gamma, colony stimulating factors (CSFs), M-CSF, GM-CSF, G-CSF, interleukins (ILs), IL-1, IL-2, IL-3, IL-4, superoxide dismutase, decay accelerating factor, viral antigen, HIV envelope proteins, GP 120, GP 140, atrial natriuretic peptide A, atrial natriuretic peptide B, atrial natriuretic peptide C, immunoglobulins, and derivatives thereof.
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11. A method for selecting novel polypeptides comprising:
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(a) constructing a replicable expression vector comprising a transcription regulatory element operably linked to a gene fusion, wherein the gene fusion comprises; (i) a first gene encoding a polypeptide; (ii) a second gene encoding a substrate peptide; and (iii) a third gene encoding at least a portion of a phage coat protein, wherein the 3'"'"' end of the first gene is linked to the 5'"'"' end of the second gene, and the 3'"'"' end of the second gene is linked to the 5'"'"' end of the third gene; b) mutating the vector at one or more selected positions within the second gene thereby forming a family of related plasmids encoding substrate peptides; (c) transforming suitable host cells with the plasmids; (d) infecting the transformed host cells with a helper phage having a gene encoding the phage coat protein; (e) culturing the transformed infected host cells under conditions suitable for forming recombinant phagemid particles containing at least a portion of the plasmid and capable of transforming the host, the conditions adjusted so that no more than a minor amount of phagemid particles display more than one copy of the fusion protein on the surface of the particle; (f) exposing the phagemid particles to at least one protease; (g) derivatizing the polypeptide with a substituent capable of binding with an affinity molecule; (h) contacting the family of exposed derivitized particles with an affinity molecule, wherein the affinity molecule has affinity for the substituent; and (i) separating the phagemid particle that bind to the affinity molecule from those that do not. - View Dependent Claims (12, 19, 20, 24, 25)
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21. A method for selecting novel polypeptides, comprising the steps of:
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(a) generating a family of related plasmids encoding substrate peptides which differ at one or more amino acid residues, the plasmids comprising a transcription regulatory element operably linked to a gene fusion, wherein the gene fusion comprises; (i) a first gene encoding a polypeptide; (ii) a second gene encoding a substrate peptide; (iii) a third gene encoding at least a portion of a phage coat protein, wherein the 3'"'"' end of the first gene is linked to the 5'"'"' end of the second gene, and the 3'"'"' end of the second gene is linked to the 5'"'"' end of the third gene; (b) transforming suitable host cells with the plasmids; (c) infecting the transformed host cells with a helper phage having a gene encoding the phage coat protein; (d) culturing the transformed infected host cells under conditions suitable for forming recombinant phagemid particles containing at least a portion of the plasmid and capable of transforming the host, the conditions adjusted so that no more than a minor amount of phagemid particles display more than one copy of the fusion protein on the surface of the particle; (e) exposing the phagemid particles to at least one protease; (f) contacting the protease treated phagemid particles with an affinity molecule, wherein the affinity molecule has affinity for the polypeptide encoded by the first gene; and (g) separating the phagemid particles that bind to the affinity molecules from those that do not bind. - View Dependent Claims (22, 23)
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Specification