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Methods for protein interaction determination

  • US 20050164214A1
  • Filed: 05/10/2004
  • Published: 07/28/2005
  • Est. Priority Date: 05/09/2003
  • Status: Active Grant
First Claim
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1. A method for identifying a plurality of pairs of interacting proteins wherein a pair of interacting proteins comprises a first test protein and a second test protein, wherein the first and second test proteins interact with each other in a cell, the method comprising the steps of:

  • a) providing a cDNA library;

    b) providing a plurality of a first plasmid comprising a coding sequence for a DNA binding domain of a transcription activator, a first recombinase recognition site, a first selectable marker, a first Type II S restriction site and a first inserted cDNA encoding a first test protein;

    c) providing a plurality of a second plasmid comprising a coding sequence for a transcription activation domain of the transcription activator, a second recombinase recognition site, a second selectable marker and a second Type II S restriction site, and a second inserted cDNA encoding a second test protein, wherein the first and second recombinase recognition sites may be identical or distinct and the first and second Type II S restriction sites may be identical or distinct;

    d) introducing the first and second plasmids from b) and c) into the same cell;

    e) inducing the expression of the recombinase to recombine the first and second introduced plasmids;

    f) isolating and digesting the recombined plasmids with a Type II S restriction enzyme to obtain restriction fragments and ligating the restriction fragments to a universal adapter to provide a pool of digested fragments flanked by universal adapter sequences;

    g) forming concatamers from the pool of digested fragments and sequencing the concatamers to determine the identity of the plurality of pairs of interacting proteins.

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