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Cell-free synthesis and isolation of novel genes and polypeptides

  • US 5,658,754 A
  • Filed: 11/29/1991
  • Issued: 08/19/1997
  • Est. Priority Date: 10/05/1989
  • Status: Expired due to Term
First Claim
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1. A method for producing novel polypeptides, comprising:

  • (a) constructing an in vitro expression unit comprising a 5'"'"' untranslated region containing an RNA polymerase binding site, a ribosome binding site, and a translation initiation signal, said expression unit being capable of producing mRNA;

    (b) attaching one or more semi-random polynucleotides to said expression unit;

    (c) transcribing or replicating from the RNA polymerase binding site the polynucleotides associated with the expression unit and semi-random polynucleotides to produce mRNA;

    (d) translating said mRNA to produce polysomes under conditions sufficient to maintain said polysomes;

    (e) binding said polysomes to a substance of interest;

    (f) isolating said polysomes that bind to said substance of interest;

    (g) disrupting said isolated polysomes to release mRNA;

    (h) recovering said mRNA;

    (i) constructing cDNA from said recovered mRNA; and

    (j) expressing said cDNA to produce novel polypeptides.

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