Details for Patent: 5,780,279

Title:	Method of selection of proteolytic cleavage sites by directed evolution and phagemid display
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.
Inventor(s):	Matthews; David J. (San Francisco, CA), Wells; James A. (Burlingame, CA), Zoller; Mark J. (San Francisco, CA)
Assignee:	Genentech, Inc. (South San Francisco, CA)
Filing Date:	Apr 05, 1995
Application Number:	08/418,928
Claims:	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. 2. The method of claim 1 further comprising determining the rate of proteolytic hydrolysis of those particles that do not bind. 3. The method of claim 2 further comprising determining the site of hydrolysis. 4. The method of claim 3 further comprising determining the sequence surrounding the site of hydrolysis. 5. The method of claim 1 wherein the phagemid particles that bind to the affinity molecule are eluted, infected into suitable host cells, and steps (d) through (h) are repeated two or more times. 6. The method of claim 1 wherein the phagemid particles that do not bind to the affinity molecule are infected into suitable host cells and steps (d) through (h) are repeated two or more times. 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. 8. The method of claim 1 wherein the first gene is mutated at one or more selected positions. 9. The method of claim 1 wherein the protease is selected from the group consisting of; HIV protease, thrombin, human neutrophil elastase and other serine proteases, Factor Xa, tissue plasminogen activator (t-PA), urokinase, streptokinase, angiotensin converting enzyme and other metalloproteases, prohormone converting enzymes selected from pancreatic protease and enterokinase, and sub tilisin. 10. The method of claim 2 wherein the substrate peptide sequence is Ala-Ala-His-Tyr-Thr-Arg-Gln (SEQ ID NO. 83). 11. 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; (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. 12. The method of claim 11 wherein step (g) is carried out prior to step (f). 13. The method of claim 1, wherein the substrate peptide comprises 3-10 amino acids. 14. The method of claim 1, wherein the replicable expression vector comprises a promoter selected from the group consisting of lacZ, alkaline phosphatase pho A, bacteriophage APL, tac, tryptophan, and bacteriophage T7 promoters. 15. The method of claim 1, wherein the culturing step forms recombinant phagemid particles wherein fewer than about 1% of the phagemid particles contain multiple copies of the fusion protein. 16. The method of claim 1, wherein the culturing step forms recombinant phagemid particles wherein the number of fusion proteins per phagemid particle is about 0.1 (number of bulk fusion proteins/number of phagemid particles). 17. The method of claim 1, wherein the phage is M13, the coat protein is M13 gene III coat protein and the host is E. coli. 18. The method of claim 17, wherein the helper phage is M13KO7. 19. The method of claim 11, wherein the phage is M13, the coat protein is M13 gene III coat protein and the host is E. coli. 20. The method of claim 19, wherein the helper phage is M13KO7. 21. A method for selecting novel polypeptides, comprising the steps of: (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. 22. The method of claim 21 wherein the phage is M13, the coat protein is M13 gene III coat protein, and the host is E. coli. 23. The method of claim 22, wherein the helper phage is M13KO7. 24. The method of claim 19, wherein the culturing step forms recombinant phagemid particles wherein fewer than about 1% of the phagemid particles contain multiple copies of the fusion protein. 25. The method of claim 19, wherein the culturing step forms phagemid particles wherein the number of fusion proteins per phagemid particle is about 0.1 (number of bulk fusion proteins/number of phagemid particles).