224
even a protein that shuttles between the cytosol and the nucleus.
PPI- induced reconstituted TEV protease activity cleaves off GV,
which translocates into the nucleus and activates a firefly luciferase
reporter gene, which is placed under the control of upstream activating sequences that contain DNA sequences recognized by GV.
Split TEV assays can be used to monitor ligand-dependent
interactions of cell surface receptors, such as RTKs and GPCRs,
thus allowing to assess their activity. For instance, the Neuregulin1induced association between ERBB4, an RTK of the ERBB family,
and the regulatory subunit alpha of the PI3K, PIK3R1, was determined in a dose-response assay (Fig. 3a), as well as its inhibition by
lapatinib (Fig. 3b). Sample constitutive interactions assayed using
split TEV are shown for the membrane-associated protein KIBRA
(Fig. 3c). For assessing GPCR activities, we have recently reported
a detailed protocol on the split TEV-based GPCR/β-Arrestin2
recruitment assay, which uses a truncated version of β-Arrestin2
and a modified NTEV-tevS-GV tag for improved sensitivity [38].
We therefore refer the reader to this specified GPCR split TEV
protocol. Notably, the split TEV method proved to be more sensitive than the full-TEV Tango approach when assaying selected
GPCR activities [15].
Interactions between candidate proteins that stay strictly cytosolic and do not shuttle into the nucleus may be monitored using
the hybrid NTEV-tevS-GV tag (see Note 1). For candidate proteins that show a certain degree of nuclear localization, candidates are fused to the NTEV fragment only (Fig. 2b). The other
candidate protein is fused to CTEV. When pursuing the sole
NTEV/CTEV tagging strategy, PPI-induced TEV protease activity leads to the activation of a cytosolically localized transcriptional TEV reporter (denoted GV-2ER), which contains a central
GV unit that is trapped by two ERT2 domains, each fused to GV
via a tevS. ERT2 domains are modified oestrogen receptor
domains, that do not respond to endogenous oestrogen, but to
4-hydroxytamoxifen. Liberated GV translocates into the nucleus
and activates the firefly luciferase reporter. MST1 dimer formation based on the C-terminal SARAH domain is shown as an
example for soluble protein interactions (Fig. 3d). Dynamic
interactions like AKT-induced and phosphorylation-regulated
BAD/14–3-3 and rapamycin- regulated FKBP/FRB have also
been described using split TEV [9, 33].
When studying PPIs using split TEV, tagged proteins of interest
are commonly introduced into cells at rather high expression levels, either transiently by transfection or by stable integration.
Therefore, assay components should be expressed at rather low
levels, possibly close to those of endogenous counterparts.
Further, each assay should be individually validated by specific
1.3 Split TEV Assays
for Soluble Proteins
1.4 Assay Controls
Jan P. Wintgens et al.
even a protein that shuttles between the cytosol and the nucleus.
PPI- induced reconstituted TEV protease activity cleaves off GV,
which translocates into the nucleus and activates a firefly luciferase
reporter gene, which is placed under the control of upstream activating sequences that contain DNA sequences recognized by GV.
Split TEV assays can be used to monitor ligand-dependent
interactions of cell surface receptors, such as RTKs and GPCRs,
thus allowing to assess their activity. For instance, the Neuregulin1induced association between ERBB4, an RTK of the ERBB family,
and the regulatory subunit alpha of the PI3K, PIK3R1, was determined in a dose-response assay (Fig. 3a), as well as its inhibition by
lapatinib (Fig. 3b). Sample constitutive interactions assayed using
split TEV are shown for the membrane-associated protein KIBRA
(Fig. 3c). For assessing GPCR activities, we have recently reported
a detailed protocol on the split TEV-based GPCR/β-Arrestin2
recruitment assay, which uses a truncated version of β-Arrestin2
and a modified NTEV-tevS-GV tag for improved sensitivity [38].
We therefore refer the reader to this specified GPCR split TEV
protocol. Notably, the split TEV method proved to be more sensitive than the full-TEV Tango approach when assaying selected
GPCR activities [15].
Interactions between candidate proteins that stay strictly cytosolic and do not shuttle into the nucleus may be monitored using
the hybrid NTEV-tevS-GV tag (see Note 1). For candidate proteins that show a certain degree of nuclear localization, candidates are fused to the NTEV fragment only (Fig. 2b). The other
candidate protein is fused to CTEV. When pursuing the sole
NTEV/CTEV tagging strategy, PPI-induced TEV protease activity leads to the activation of a cytosolically localized transcriptional TEV reporter (denoted GV-2ER), which contains a central
GV unit that is trapped by two ERT2 domains, each fused to GV
via a tevS. ERT2 domains are modified oestrogen receptor
domains, that do not respond to endogenous oestrogen, but to
4-hydroxytamoxifen. Liberated GV translocates into the nucleus
and activates the firefly luciferase reporter. MST1 dimer formation based on the C-terminal SARAH domain is shown as an
example for soluble protein interactions (Fig. 3d). Dynamic
interactions like AKT-induced and phosphorylation-regulated
BAD/14–3-3 and rapamycin- regulated FKBP/FRB have also
been described using split TEV [9, 33].
When studying PPIs using split TEV, tagged proteins of interest
are commonly introduced into cells at rather high expression levels, either transiently by transfection or by stable integration.
Therefore, assay components should be expressed at rather low
levels, possibly close to those of endogenous counterparts.
Further, each assay should be individually validated by specific
1.3 Split TEV Assays
for Soluble Proteins
1.4 Assay Controls
Jan P. Wintgens et al.