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Rational evolution of cytokines for higher stability, the cytokines and encoding nucleic acid molecules

  • US 7,650,243 B2
  • Filed: 02/14/2007
  • Issued: 01/19/2010
  • Est. Priority Date: 09/09/2002
  • Status: Expired due to Fees
First Claim
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1. A method for generating a cytokine having a predetermined property or activity, the method comprising:

  • (a) identifying, within a target cytokine or plurality thereof, target amino acids, wherein;

    each target amino acid is designated an in silico-HIT (is-HIT); and

    the is-HIT target amino acids are identified by identifying structurally homologous loci between the target cytokine and a reference cytokine possessing a predetermined activity, wherein;

    the predetermined property or activity is resistance to proteolysis; and

    identification of the structurally homologous loci between the target cytokine and the reference cytokine possessing the predetermined activity is effected by;

    (i) comparing the 3-dimensional structures of the two or more proteins to identify regions of high coincidence between their backbones, said regions designated as structurally homologous regions, wherein the degree of coincidence between the 3-dimensional structures of the target cytokine and of the reference cytokine is determined by superposition and root means squared (RMS) deviation calculations using any combination of one or more of the peptide backbone atoms selected from the group consisting of;

    N, C, C(C═

    O), O and CA; and

    (ii) identifying is-HIT structurally homologous loci on the target cytokine that correspond to structurally related is-HIT amino acid positions within a structurally homologous region of the reference cytokine;

    (b) identifying one or more replacement amino acids, specific for each is-HIT, wherein;

    each cytokine comprising a single amino acid replacement within the target cytokine is designated as a candidate LEAD protein; and

    the replacement amino acids are selected using Percent Accepted Mutations (PAM) matrices;

    (c) producing a population of separate sets of nucleic acid molecules, wherein;

    each set encodes a different candidate LEAD protein;

    all nucleic acid molecules in a set encode the same protein;

    each candidate LEAD protein contains a single amino acid replacement; and

    each set of nucleic acid molecules encodes a candidate LEAD protein that differs by one amino acid from the target cytokine;

    (d) separately introducing each set of nucleic acid molecules into host cells and expressing the encoded candidate LEAD proteins, whereby the host cells comprise an addressable array such that each LEAD protein is expressed at a different locus in the array, and the identity of each candidate LEAD protein at each locus is known; and

    (e) individually screening each set of encoded candidate LEAD proteins to identify one or more proteins that has an activity that differs from an activity an unmodified target cytokine wherein;

    each such cytokine is designated a LEAD mutant protein.

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