221
is induced by the regulated recruitment of G protein-coupled
receptor kinases (GRKs) that phosphorylate residues on the C terminus of the GPCR and thus provide a platform for β-Arrestin
binding [13]. The binding of β-arrestin sterically obstructs G protein coupling and triggers the internalization of the GPCR. The
regulated association between a given GPCR and β-arrestin2 can
be exploited to reliably monitor GPCR activities and is regularly
used by SBA and related proximity assays [14–17].
To assess dynamic interactions among proteins, various
approaches for genetically encoded SBA are available, including,
but not limited to, GFP and variants thereof [18–20], luciferases
(firefly [21, 22], Renilla [23, 24], Gaussia [25], click beetle luciferases [17, 26, 27]), β-lactamase [28], β-galactosidase [29, 30],
ubiquitin [8, 31, 32], and the tobacco etch virus (TEV) protease
[33]. In addition to SBA, proximity assays were developed, such as
bioluminescence resonance energy transfer (BRET) assays comprising firefly luciferase and GFP [34, 35] and the Tango method
that uses a full-length TEV protease fused to an interacting partner
like β-Arrestin to monitor GPCR activities [16].
Here, we present a detailed protocol for the split TEV technique, a genetically encoded SBA that can be flexibly applied to
sensitively monitor various types of regulated PPIs in living cells
[33] (and reviewed in [6]). The technique is based on the
interaction- induced fragment complementation of the TEV protease. Split TEV can be used to assess dynamic interactions at the
membrane and in the cytosol, thus allowing various combinations among potentially interacting candidate proteins (Fig. 1).
Notably, interactions in the nucleus cannot be monitored using
the presented technique, as the method was specifically designed
to analyze PPIs at the membrane and in the cytosol. The technique integrates various reporter systems, including fluorescent
and luminescent reporters, making it implementable to many
research laboratories. As split TEV flexibly allows using readouts
of choice, scalable transcriptional barcode reporters that are amenable to multiplexed high-throughput formats and next-generation sequencing may also be used. Combining these technologies
will enable assessing drug target activities and cellular response
profiles in parallel, thereby opening up new avenues in drug discovery. Here, we centre on luciferase reporters, as these are widely
used both in single interaction assays and in high-throughput
applications.
For split TEV assays, an optimized form of the TEV protease
is dissected into an N-terminal (NTEV, amino acids 1–118) and a
C-terminal fragment (CTEV, amino acids 119–221, with a Ser- >
Pro substitution at residue 219 for enhanced assay stability). The
S219P mutation renders the TEV protease refractory to autocatalysis [36]. In addition, the optimized form is truncated after
amino acid 221 to remove the inhibitory C-terminal tail, which
Characterising Dynamic PPIs Using Split TEV
is induced by the regulated recruitment of G protein-coupled
receptor kinases (GRKs) that phosphorylate residues on the C terminus of the GPCR and thus provide a platform for β-Arrestin
binding [13]. The binding of β-arrestin sterically obstructs G protein coupling and triggers the internalization of the GPCR. The
regulated association between a given GPCR and β-arrestin2 can
be exploited to reliably monitor GPCR activities and is regularly
used by SBA and related proximity assays [14–17].
To assess dynamic interactions among proteins, various
approaches for genetically encoded SBA are available, including,
but not limited to, GFP and variants thereof [18–20], luciferases
(firefly [21, 22], Renilla [23, 24], Gaussia [25], click beetle luciferases [17, 26, 27]), β-lactamase [28], β-galactosidase [29, 30],
ubiquitin [8, 31, 32], and the tobacco etch virus (TEV) protease
[33]. In addition to SBA, proximity assays were developed, such as
bioluminescence resonance energy transfer (BRET) assays comprising firefly luciferase and GFP [34, 35] and the Tango method
that uses a full-length TEV protease fused to an interacting partner
like β-Arrestin to monitor GPCR activities [16].
Here, we present a detailed protocol for the split TEV technique, a genetically encoded SBA that can be flexibly applied to
sensitively monitor various types of regulated PPIs in living cells
[33] (and reviewed in [6]). The technique is based on the
interaction- induced fragment complementation of the TEV protease. Split TEV can be used to assess dynamic interactions at the
membrane and in the cytosol, thus allowing various combinations among potentially interacting candidate proteins (Fig. 1).
Notably, interactions in the nucleus cannot be monitored using
the presented technique, as the method was specifically designed
to analyze PPIs at the membrane and in the cytosol. The technique integrates various reporter systems, including fluorescent
and luminescent reporters, making it implementable to many
research laboratories. As split TEV flexibly allows using readouts
of choice, scalable transcriptional barcode reporters that are amenable to multiplexed high-throughput formats and next-generation sequencing may also be used. Combining these technologies
will enable assessing drug target activities and cellular response
profiles in parallel, thereby opening up new avenues in drug discovery. Here, we centre on luciferase reporters, as these are widely
used both in single interaction assays and in high-throughput
applications.
For split TEV assays, an optimized form of the TEV protease
is dissected into an N-terminal (NTEV, amino acids 1–118) and a
C-terminal fragment (CTEV, amino acids 119–221, with a Ser- >
Pro substitution at residue 219 for enhanced assay stability). The
S219P mutation renders the TEV protease refractory to autocatalysis [36]. In addition, the optimized form is truncated after
amino acid 221 to remove the inhibitory C-terminal tail, which
Characterising Dynamic PPIs Using Split TEV