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Method of selection of proteolytic cleavage sites by directed evolution and phagemid display

  • US 5,780,279 A
  • Filed: 04/05/1995
  • Issued: 07/14/1998
  • Est. Priority Date: 12/03/1990
  • Status: Expired due to Term
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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