Detection of molecular interactions using a reduced affinity enzyme complementation reporter system

A method of determining whether a first and second protein interact by binding to each other, said method comprising:

• (a) providing a mammalian cell comprising:
• (i) a first fusion protein of said first protein and a first β-galactosidase fragment of a full length β-galactosidase enzyme, wherein said first β-galactosidase fragment is a variant minimal N-terminal β-galactosidase peptide about 60 amino acids or less in length;
• and
• (ii) a second fusion protein of said second protein and a second β-galactosidase fragment including greater than 60% of the full-length β-galactosidase enzyme;
• 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, wherein said variant minimal N-terminal β-galactosidase peptide comprises a point mutation, insertion or deletion between amino acid residues 29 to 41, or a truncation terminating between amino acid residues 48 and 43, as set forth in a β-galactosidase peptide sequence according to SEQ ID NO:
• 7;
• and
• (b) evaluating said cell for β-galactosidase activity to determine whether said first and second proteins interact by said binding to each other, thereby complementing said first β-galactosidase fragment and said second β-galactosidase fragment.