We have previously described a strategy for detecting
protein−protein interactions based on protein interaction−assisted
folding of rationally designed fragments of enzymes. We call this strategy the
protein fragment complementation assay (PCA)1,
2,
3,
4,
5. Here
we describe PCAs based on the enzyme TEM-1 -lactamase (EC: 3.5.2.6),
which include simple colorimetric in vitro assays using the
cephalosporin nitrocefin and assays in intact cells using the fluorescent
substrate CCF2/AM (ref. 6). Constitutive
protein−protein interactions of the GCN4 leucine zippers and of apoptotic
proteins Bcl2 and Bad, and the homodimerization of Smad3, were tested in an
in vitro assay using cell lysates. With the same in vitro assay,
we also demonstrate interactions of protein kinase PKB with substrate Bad. The
in vitro assay is facile and amenable to high-throughput modes of
screening with signal-to-background ratios in the range of 10:1 to 250:1, which
is superior to other PCAs developed to date. Furthermore, we show that the
in vitro assay can be used for quantitative analysis of a small
molecule−induced protein interaction, the rapamycin-induced interaction
of FKBP and yeast FRB (the FKBP-rapamycin binding domain of TOR (target of
rapamycin)). The assay reproduces the known dissociation constant and number of
sites for this interaction. The combination of in vitro colorimetric and
in vivo fluorescence assays of -lactamase in mammalian cells
suggests a wide variety of sensitive and high-throughput large-scale
applications, including in vitro protein array analysis of
protein−protein or enzyme−protein interactions and in vivo
applications such as clonal selection for cells expressing interacting protein
partners.
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-Lactamase protein fragment complementation assays as in
vivo and in vitro sensors of protein−protein
interactions
-lactamase (EC: 3.5.2.6),
which include simple colorimetric in vitro assays using the
cephalosporin nitrocefin and assays in intact cells using the fluorescent
substrate CCF2/AM (ref. 6). Constitutive
protein−protein interactions of the GCN4 leucine zippers and of apoptotic
proteins Bcl2 and Bad, and the homodimerization of Smad3, were tested in an
in vitro assay using cell lysates. With the same in vitro assay,
we also demonstrate interactions of protein kinase PKB with substrate Bad. The
in vitro assay is facile and amenable to high-throughput modes of
screening with signal-to-background ratios in the range of 10:1 to 250:1, which
is superior to other PCAs developed to date. Furthermore, we show that the
in vitro assay can be used for quantitative analysis of a small
molecule−induced protein interaction, the rapamycin-induced interaction
of FKBP and yeast FRB (the FKBP-rapamycin binding domain of TOR (target of
rapamycin)). The assay reproduces the known dissociation constant and number of
sites for this interaction. The combination of in vitro colorimetric and
in vivo fluorescence assays of 
signalling through a direct interaction with Smad3
