121
number. For this mutant, the oxidative luminescent reaction is synergistically suppressed by the H245D/K443A mutations while the
adenylation reaction is enhanced by the L530R mutation. As a
result, the Donor can efficiently produce LH 2 -AMP, but can only
poorly produce OxL. On the other hand, the Acceptor that originally featured a K529Q mutation had a lower adenylation activity
while maintaining full oxidative luminescent activity. Thus, the
Acceptor cannot produce LH 2 -AMP efficiently on its own.
To perform the interaction assay, the Donor and the Acceptor
are each fused to one of two interacting proteins of interest, respectively. When they do not interact with each other, the Donor and the
Acceptor are far apart, and the luminescence intensity remains low.
However, when they are brought into close proximity by the interacting protein pair, the Acceptor can react with LH 2 -AMP that was
produced by the Donor. As a result, the luminescence that is emitted
is increased (Fig. 1b). The splitting of two half reactions is made
possible partly due to the conformational change of the Fluc enzyme,
which accompanies rotation of the C-terminal domain.
So far, several attempts to improve the PPI assay FlimPIA have
been achieved, and several advantages over conventional PPI assays
have been discovered [1–3]. First, FlimPIA generally works well
in vitro as well as in cultured cells. Second, compared to conventional Fluc-based PPI assays based on protein-fragment complementation assays (PCA), the FlimPIA probes are more thermostable
and reproducibly give higher luminescence. Third, the distance
that can be detected between the probes is longer than the distance
between the probes for Förster/fluorescence resonance energy
transfer (FRET)-based PPI assays and PCA. In practice, this means
that larger interacting proteins can be detected by FlimPIA. Fourth,
the sensitivity is approximately tenfold higher than Fluc-based
PCA both in terms of the detection limit and signal intensity
in vitro. Finally, using a recently discovered double mutant R437K/
K529Q as a new Acceptor significantly improved signal/background
(S/B) ratio as well as the signal intensity under optimized assay
conditions. The mutation of residue R437 which is located in the
hinge region connecting the N-terminal and C-terminal domains
was found to selectively lower adenylation activity. The obtained
maximum S/B ratio (> 40) is higher than that obtained with Fluc
PCA under the same experimental reaction conditions. In this
chapter, a protocol to generate and assay this latest version of
FlimPIA is described [2].
2 Materials
Prepare all solutions using ultrapure water (made by purifying
deionized water to attain resistivity of 18 MΩ cm at 25 °C). For all
the reagents, use them of the highest grade available.
Sensitive Protein-Protein Interaction Assay
number. For this mutant, the oxidative luminescent reaction is synergistically suppressed by the H245D/K443A mutations while the
adenylation reaction is enhanced by the L530R mutation. As a
result, the Donor can efficiently produce LH 2 -AMP, but can only
poorly produce OxL. On the other hand, the Acceptor that originally featured a K529Q mutation had a lower adenylation activity
while maintaining full oxidative luminescent activity. Thus, the
Acceptor cannot produce LH 2 -AMP efficiently on its own.
To perform the interaction assay, the Donor and the Acceptor
are each fused to one of two interacting proteins of interest, respectively. When they do not interact with each other, the Donor and the
Acceptor are far apart, and the luminescence intensity remains low.
However, when they are brought into close proximity by the interacting protein pair, the Acceptor can react with LH 2 -AMP that was
produced by the Donor. As a result, the luminescence that is emitted
is increased (Fig. 1b). The splitting of two half reactions is made
possible partly due to the conformational change of the Fluc enzyme,
which accompanies rotation of the C-terminal domain.
So far, several attempts to improve the PPI assay FlimPIA have
been achieved, and several advantages over conventional PPI assays
have been discovered [1–3]. First, FlimPIA generally works well
in vitro as well as in cultured cells. Second, compared to conventional Fluc-based PPI assays based on protein-fragment complementation assays (PCA), the FlimPIA probes are more thermostable
and reproducibly give higher luminescence. Third, the distance
that can be detected between the probes is longer than the distance
between the probes for Förster/fluorescence resonance energy
transfer (FRET)-based PPI assays and PCA. In practice, this means
that larger interacting proteins can be detected by FlimPIA. Fourth,
the sensitivity is approximately tenfold higher than Fluc-based
PCA both in terms of the detection limit and signal intensity
in vitro. Finally, using a recently discovered double mutant R437K/
K529Q as a new Acceptor significantly improved signal/background
(S/B) ratio as well as the signal intensity under optimized assay
conditions. The mutation of residue R437 which is located in the
hinge region connecting the N-terminal and C-terminal domains
was found to selectively lower adenylation activity. The obtained
maximum S/B ratio (> 40) is higher than that obtained with Fluc
PCA under the same experimental reaction conditions. In this
chapter, a protocol to generate and assay this latest version of
FlimPIA is described [2].
2 Materials
Prepare all solutions using ultrapure water (made by purifying
deionized water to attain resistivity of 18 MΩ cm at 25 °C). For all
the reagents, use them of the highest grade available.
Sensitive Protein-Protein Interaction Assay