227
interactions between full-length candidate proteins in living cells
or in lysates thereof, (2) the detection of protein interactions
localized at the membrane, sub-membrane compartment, and the
cytosol, (3) the detection of protein interactions in heterologous
mammalian cells, primary cell types including primary neurons
and astrocytes, (4) the detection of regulated RTK and GPCR
activities induced by extracellular stimuli, (5) the analysis of regulated, ligand or compound concentration-dependent interactions
in dose-response assays assessing actions of agonists and antagonists, and (6) the setup as protein interaction readouts for highthroughput screening (HTS) purposes [9, 15, 33, 40–42].
Therefore, split TEV may also be adapted to industrial HTS setups to screen for RTK and GPCR activities, for example in drug
discovery programs.
Below, we describe experimental setups for split TEV assays for
membrane, membrane-associate and cytosolic proteins using firefly
luciferase as a final reporter gene as this readout is widely used
across laboratories, and allows adaptation to HTS approaches.
2 Materials
1. For membrane, membrane-associated and strictly cytosolically
localized proteins:
ORF(X)-NTEV-tevS-GV plasmid. The ORF of candidate X is
fused to NTEV (amino acids 1–118), tevS (ENLYFQG),
and GV (Fig. 2a). Use molecular cloning to generate candidate X fusion vectors (see Note 2). For a detailed description on GPCRs, see the protocol by Wehr et al. [40].
For cytosolic protein candidates that shuffle between cytosol
and nucleus:
ORF(X)-NTEV plasmid. The ORF of candidate X is fused to
NTEV (amino acids 1–118) only. In addition, the pGV2ER plasmid is required as cytosolic TEV reporter (Fig. 2b).
2. ORF(Y)-CTEV. The ORF of candidate Y is fused to CTEV
(amino acids 119–221, including a S219P mutation). Use
molecular cloning to generate the fusion vector (see Note 2).
3. Firefly luciferase reporter plasmid. Use an upstream activating
enhancer sequences (UAS)-driven firefly luciferase (e.g.,
pGL4.31[luc2P/GAL4UAS/Hygro], Promega, catalog no.
C9351) (see Note 3).
4. Renilla luciferase control plasmid. Use a thymidine kinase
(TK)-driven constitutive Renilla luciferase as internal control
(pRL-TK, Promega, catalog no. E2241).
5. A GFP or GFP-derivative expressing plasmid for optical transfection control (e.g., mVenus-N1, Addgene, Plasmid #54640).
2.1 Plasmids
Characterising Dynamic PPIs Using Split TEV
interactions between full-length candidate proteins in living cells
or in lysates thereof, (2) the detection of protein interactions
localized at the membrane, sub-membrane compartment, and the
cytosol, (3) the detection of protein interactions in heterologous
mammalian cells, primary cell types including primary neurons
and astrocytes, (4) the detection of regulated RTK and GPCR
activities induced by extracellular stimuli, (5) the analysis of regulated, ligand or compound concentration-dependent interactions
in dose-response assays assessing actions of agonists and antagonists, and (6) the setup as protein interaction readouts for highthroughput screening (HTS) purposes [9, 15, 33, 40–42].
Therefore, split TEV may also be adapted to industrial HTS setups to screen for RTK and GPCR activities, for example in drug
discovery programs.
Below, we describe experimental setups for split TEV assays for
membrane, membrane-associate and cytosolic proteins using firefly
luciferase as a final reporter gene as this readout is widely used
across laboratories, and allows adaptation to HTS approaches.
2 Materials
1. For membrane, membrane-associated and strictly cytosolically
localized proteins:
ORF(X)-NTEV-tevS-GV plasmid. The ORF of candidate X is
fused to NTEV (amino acids 1–118), tevS (ENLYFQG),
and GV (Fig. 2a). Use molecular cloning to generate candidate X fusion vectors (see Note 2). For a detailed description on GPCRs, see the protocol by Wehr et al. [40].
For cytosolic protein candidates that shuffle between cytosol
and nucleus:
ORF(X)-NTEV plasmid. The ORF of candidate X is fused to
NTEV (amino acids 1–118) only. In addition, the pGV2ER plasmid is required as cytosolic TEV reporter (Fig. 2b).
2. ORF(Y)-CTEV. The ORF of candidate Y is fused to CTEV
(amino acids 119–221, including a S219P mutation). Use
molecular cloning to generate the fusion vector (see Note 2).
3. Firefly luciferase reporter plasmid. Use an upstream activating
enhancer sequences (UAS)-driven firefly luciferase (e.g.,
pGL4.31[luc2P/GAL4UAS/Hygro], Promega, catalog no.
C9351) (see Note 3).
4. Renilla luciferase control plasmid. Use a thymidine kinase
(TK)-driven constitutive Renilla luciferase as internal control
(pRL-TK, Promega, catalog no. E2241).
5. A GFP or GFP-derivative expressing plasmid for optical transfection control (e.g., mVenus-N1, Addgene, Plasmid #54640).
2.1 Plasmids
Characterising Dynamic PPIs Using Split TEV