Protein fragment complementation assays for the detection of biological or drug interactions

A method for detecting biomolecular interactions said method comprising:

• (a) selecting an appropriate enzyme reporter molecule;
• (b) effecting fragmentation of said enzyme reporter molecule such that said fragmentation results in reversible loss of reporter function;
• (c) fusing or attaching fragments of said enzyme reporter molecule separately to other molecules;
• (d) reassociating said enzyme reporter fragments through interactions of the molecules that are fused or attached to said fragments;
• and
• (e) detecting the activity of said enzyme reporter molecule.

A method of testing biomolecular interactions comprising:

• a) generating a first fusion product comprising:
• i) a first fragment of a first enzyme molecule and
• ii) a second molecule which is different from said first molecule;
• b) generating a second fusion product comprising
• i) a second fragment of said first enzyme molecule;
• and
• ii) a third molecule which is different from said first molecule or second molecule;
• c) allowing the first and second fusion products to contact each other;
• and
• d) testing for activity regained by association of the enzyme fragments of the first molecule, wherein said reassociation is mediated by interaction of the second and third molecules.

A composition comprising a product selected from the group consisting of:

• (a) a first fusion product comprising:
• 1) a first fragment of a first enzyme molecule whose fragments exhibit a detectable activity when associated and
• 2) a second molecule that is fused to said first fragment;
• (b) a second fusion product comprising
• 1) a second fragment of said first enzyme molecule and
• 2) a third molecule that is fused to said second fragment;
• and
• c) both (a) and (b).

A composition comprising a nucleic acid molecule coding for an enzyme fusion product, which molecule comprises sequences coding for a product selected from the group consisting of:

• (a) a first enzyme fusion product cornprising:
• 1) fragments of a first enzyme molecule whose fragments can exhibit a detectable activity when associated and
• 2) a second molecule fused to the fragment of the first molecule;
• (b) a second fusion product comprising
• 1) a second fragment of said first enzyme molecule and
• 2) a second or third molecule;
• and
• (c) both (a) and (b).

A method of testing for biomolecular interactions associated with:

• (a) complementary fragments of a first enzyme molecule whose fragments can exhibit a detectable activity when associated, said method comprising:
• 1) creating a fusion of
• (a) a first fragment of a first enzyme molecule whose fragments can exhibit a detectable activity when associated and
• (b) a first protein-protein interacting domain;
• 2) creating a fusion of
• (a) a second fragment of said first enzyme molecule and
• (b) a second or third protein-protein interacting domain that can bind said first protein-protein interacting domain;
• 3) allowing the fusions of (1) and (2) to contact each other;
• and
• 4) testing for reconstitution of said enzyme activity.

A composition comprising a product selected from the group consisting of:

• (a) a first fusion product comprising:
• 1) a first fragment of an enzyme molecule whose fragments exhibit a detectable activity when associated and
• 2) a first protein-protein interacting domain;
• (b) a second fusion product comprising
• 1) a second fragment of said first enzyme molecule and
• 2) a second protein-protein interacting domain that can bind said first protein-protein interacting domain;
• and
• (c) both (a) and (b).

A composition comprising a nucleic acid molecule coding for an enzyme fusion product, which molecule comprises sequences coding for either:

• (a) a first fusion product comprising:
• 1) a first fragment of an enzyme molecule whose fragments exhibit a detectable activity when associated and
• 2) a first protein-protein interacting domain;
• or
• (b) a second fusion product comprising
• 1) a second fragment of said enzyme molecule and
• 2) a second protein-protein interacting domain that can bind said first protein-protein interacting domain;
• or
• (c) both (a) and (b).

A method for detecting protein-protein interactions in living organisms and or cells, which method comprises:

• (a) synthesizing probe protein fragments from an enzyme which enables dominant selection by dissecting the gene coding for the enzyme into at least two fragments;
• (b) constructing fusion proteins consisting of the probe protein fragments linked to protein domains that are to be tested for interactions;
• (c) coexpressing the fusion proteins;
• and
• (d) detecting reconstitution of enzyme activity.

A method for detecting biomolecular interactions said method comprising:

• (a) selecting an appropriate enzyme reporter molecule;
• (b) effecting fragmentation of said enzyme reporter molecule;
• (c) fusing or attaching fragments of said enzyme reporter molecule separately to other molecules;
• followed by
• (d) reassociation of said reporter fragments through interactions of the molecules that are fused to said fragments;
• and
• (e) detecting said biomolecular interactions by reconstitution of enzyme activity.

A method for detecting biomolecular interactions said method comprising:

• (a) selecting an appropriate reporter molecule selected from the group consisting of a multimeric enzyme, a fluorescent protein, a luminescent protein and a phosphorescent protein;
• (b) effecting fragmentation of said reporter molecule such that said fragmentation results in reversible loss of reporter function;
• (c) fusing or attaching fragments of said reporter molecule separately to other molecules;
• followed by
• (d) reassociation of said reporter fragments through interactions of the molecules that are fused to said fragments;
• and
• (e) detecting said biomolecular interactions by reconstitution of activity of the reporter molecule.