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US 2014/0178913 A1Published application
Detection of Molecular Interactions Using a Reduced Affinity Enzyme Complementation Reporter System
OwnerTom Wehrman, Helen M. Blauinventors
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Abstract
Methods and compositions for detecting molecular interactions are provided. Aspects of the invention include the use of a reduced affinity enzyme complementation reporter system. Also provided are systems and kits for use in practicing embodiments of the methods.
Claims25
A method of determining whether a first and second protein interact, said method comprising:
- (a) providing a cell comprising:
- (i) a first fusion protein of said first protein and a first β-galactosidase fragment, wherein said first β-galactosidase fragment is a variant minimal N-terminal β-galactosidase peptide;
- and
- (ii) a second fusion protein of said second protein and a second β-galactosidase fragment;
- wherein said first and second β-galactosidase fragments have an affinity for each other which provides a known level of β-galactosidase activity in the absence of an interaction between said first and second proteins that is lower than the activity observed in the presence of an interaction between said first and second proteins;
- and
- (b) evaluating said cell for β-galactosidase activity to determine whether said first and second proteins interact.
4 more elements
A cell comprising:
- (a) a first fusion protein of a first protein and a first β-galactosidase fragment, wherein said first β-galactosidase fragment is a variant minimal N-terminal β-galactosidase peptide;
- and
- (b) a second fusion protein of a second protein and a second β-galactosidase fragment;
- wherein said first and second β-galactosidase fragments have a low affinity for each other that provides a known level of β-galactosidase activity in the absence of an interaction between said first and second proteins that is lower than the activity observed in the presence of an interaction between said first and second proteins.
1 more element
A kit comprising:
- (a) cell comprising:
- (i) a first fusion protein of a first protein and a first β-galactosidase fragment, wherein said first β-galactosidase fragment is a variant minimal N-terminal β-galactosidase peptide;
- and
- (ii) a second fusion protein of said second protein and a second β-galactosidase fragment;
- wherein said first and second β-galactosidase fragments have a low affinity for each other which provides a known level of β-galactosidase activity in the absence of an interaction between said first and second proteins that is lower than the activity observed in the presence of an interaction between said first and second proteins;
- and
- (b) a β-galactosidase substrate.
4 more elements
A kit comprising:
- (a) a first nucleic acid encoding a first β-galactosidase fragment;
- and
- (b) a second nucleic acid encoding a second β-galactosidase fragment;
- wherein said first β-galactosidase fragment is a variant minimal N-terminal β-galactosidase peptide and has a binding affinity for said second β-galactosidase fragment that is lower than a β-galactosidase fragment consisting of amino acids 3 to 92 of E. coli wild-type β-galactosidase.
1 more element
Dependencies are inferred from the claim text.