232
4 Notes
1. When opting for the hybrid NTEV-tevS-GV tag, we recommend to first perform control assays that only contain the candidate-NTEV-tevS-GV to exclude high background readings.
For example, activated Sterile20-like kinase MST1 (STK4) is
cleaved [43] and produces high background readings, thus
precluding the use of the hybrid tag. In a next step, the candidate may be tested with a protein that is known not to bind to
the candidate to validate assay robustness, and to establish
experimental parameters for a reference negative control.
2. Cloning of candidate split TEV fusions. Clone the candidate
ORFs including all split TEV assay elements into a regular
expression vector, such as pcDNA3.1. The fusions are constructed to contain a 10 amino acid flexible linker (GGGGS
GGGGS) between the candidate protein and NTEV or CTEV.
As expression plasmids, we regularly use pcDNA3- derived
(Invitrogen) and pTag-derived (Stratagene) vectors. If
required, N- and CTEV fusions can be stably integrated into
the cells using the mammalian selection markers present on
these plasmids (i.e., neomycin or zeocin resistance). We regularly use Gateway recombination cloning (Life Technologies)
to quickly obtain candidate fusions. Gateway-compatible ORFs
for more than 16,000 ORFs are available at Plasmid ID
(http://plasmid.med.harvard.edu/PLASMID/Home.jsp).
The TEV protease mutant S219P can be obtained from
Addgene (plasmid no. 8830, pRK792).
3. When opting for fluorescence-based assays, use a UAS-EGFP
plasmid (i.e., pJFRC7-20XUAS-IVS-mCD8::GFP, Addgene,
plasmid no. 26220). In this case, normalization can be done
using nuclear staining or by co-transfecting a constitutively
expressing mCherry plasmid. In addition, a fluorescence plate
reader is required for quantification.
4. For investigating membrane protein interactions, we start testing assays in PC12 Tet-Off cells (Clontech) and then proceed
to HEK293 cells (ATCC). For membrane-associated and soluble proteins, we commonly start with HEK293 cells. Notably,
U2OS cells proved to a valuable cell line to monitor GPCR
activities. As we observed substantial differences in the activation ratios for some GPCRS, like AVPR1 and DRD2, and
RTKs, like ERBB family receptors, we suggest to assess the
optimal cell line for each individual assay.
5. Positive (known interacting protein) and negative (known
non-interacting protein) controls should always be included in
protein–protein interaction assays based on protein fragment
complementation [33]. As a starting guideline, we suggest
Jan P. Wintgens et al.
4 Notes
1. When opting for the hybrid NTEV-tevS-GV tag, we recommend to first perform control assays that only contain the candidate-NTEV-tevS-GV to exclude high background readings.
For example, activated Sterile20-like kinase MST1 (STK4) is
cleaved [43] and produces high background readings, thus
precluding the use of the hybrid tag. In a next step, the candidate may be tested with a protein that is known not to bind to
the candidate to validate assay robustness, and to establish
experimental parameters for a reference negative control.
2. Cloning of candidate split TEV fusions. Clone the candidate
ORFs including all split TEV assay elements into a regular
expression vector, such as pcDNA3.1. The fusions are constructed to contain a 10 amino acid flexible linker (GGGGS
GGGGS) between the candidate protein and NTEV or CTEV.
As expression plasmids, we regularly use pcDNA3- derived
(Invitrogen) and pTag-derived (Stratagene) vectors. If
required, N- and CTEV fusions can be stably integrated into
the cells using the mammalian selection markers present on
these plasmids (i.e., neomycin or zeocin resistance). We regularly use Gateway recombination cloning (Life Technologies)
to quickly obtain candidate fusions. Gateway-compatible ORFs
for more than 16,000 ORFs are available at Plasmid ID
(http://plasmid.med.harvard.edu/PLASMID/Home.jsp).
The TEV protease mutant S219P can be obtained from
Addgene (plasmid no. 8830, pRK792).
3. When opting for fluorescence-based assays, use a UAS-EGFP
plasmid (i.e., pJFRC7-20XUAS-IVS-mCD8::GFP, Addgene,
plasmid no. 26220). In this case, normalization can be done
using nuclear staining or by co-transfecting a constitutively
expressing mCherry plasmid. In addition, a fluorescence plate
reader is required for quantification.
4. For investigating membrane protein interactions, we start testing assays in PC12 Tet-Off cells (Clontech) and then proceed
to HEK293 cells (ATCC). For membrane-associated and soluble proteins, we commonly start with HEK293 cells. Notably,
U2OS cells proved to a valuable cell line to monitor GPCR
activities. As we observed substantial differences in the activation ratios for some GPCRS, like AVPR1 and DRD2, and
RTKs, like ERBB family receptors, we suggest to assess the
optimal cell line for each individual assay.
5. Positive (known interacting protein) and negative (known
non-interacting protein) controls should always be included in
protein–protein interaction assays based on protein fragment
complementation [33]. As a starting guideline, we suggest
Jan P. Wintgens et al.