Patent ID: 6365347
Filing Date: 2002-04-02
Classification: C12N

Abstract:
A method for identifying from a library of fusion macromolecules a fusion macromolecule of interest which interacts within a cell with an intracellular target molecule and detecting whether the interaction disrupts a biological pathway, wherein the cell survives or proliferates when the interaction disrupts the biological pathway and the cell dies or fails to proliferate when the interaction does not disrupt the biological pathway, and wherein the macromolecule of interest comprises a peptide library region which is presented in a restricted conformation by a heterologous carrier region, the method comprising:a) generating a panel of nucleic acid sequences that encodes a peptide library, the panel including a nucleic acid sequence that encodes the peptide library region that interacts with the intracellular target molecule within the cell. b) inserting the panel of nucleic acid sequences so generated into a plurality of vectors, each vector comprising a carrier region so as to generate an expression library of chimeric vectors. each of the chimeric vectors having a nucleic acid sequence from the panel and a nucleic acid sequence encoding the carrier region having the restricted conformation; c) introducing the expression library of chimeric vectors into a plurality of cells that lack a recombinant reporter gene so as to generate a library of cells; d) growing the library of cells so generated under suitable conditions so as to produce a library of fusion macromolecules within the cells, wherein the library of fusion macromolecules comprises the peptide library region presented in a restricted conformation by the carrier region; and e) selecting from the library of cells so grown a desired cell that produces the macromolecule of interest by detecting the interaction between the peptide library region and the target molecule which results in disruption of the biological pathway within the desired cell and enables the desired cell to survive or proliferate, thereby identifying the macromolecule produced by the cell so selected.