Snap tag sequence

The generation of NLuc-A₃-receptor (Stoddart et al., 2015 (link)) and SNAP-A₃-receptor (Stoddart et al., 2014 (link)) pcDNA3.1 plasmids has been described previously. We generated NLuc-labeled adenosine receptor constructs by amplifying the full length sequence of NLuc luciferase (as provided by Promega Corporation in the pNL1.1 vector) and fusing it in frame with the membrane signal sequence of the 5HT_3A receptor within pcDNA3.1(+) to yield sig-NLuc. We then fused the full-length human sequence of the adenosine A3-receptor (obtained from Missouri S&T cDNA Resource Centre; www.cdna.org; GenBank: AY136749) with the methionine start signal removed, to the 3′ end of the sig-NLuc in pcDNA3.1(+). This gave the construct designated as NLuc-A₃ receptor. To generate N-terminal SNAP-tagged adenosine A₃ receptor, the methionine start signal was removed from cDNA encoding the full length A₃-receptor and subcloned into a pcDNA3.1-zeo (+) vector containing the 5HT₃-receptor-derived signal sequence followed by the SNAP Tag sequence (New England Biolabs, Ipswich, MA, USA).

Mouse Junctophilin-2 cDNA (NP_001192005.1) was amplified by PCR and cloned into pGEX-6P-1 vector (GE Healthcare). Site-directed mutagenesis of Δ162-167, Δ479-486, Δ563-568 and Δ644-649 was carried out using the GeneArt™ site-directed mutagenesis kit (Thermo Fisher Scientific). For specific dye labeling, the SNAP-tag sequence (New England BioLabs) was amplified by PCR and cloned into the vector immediately downstream of the JP2 sequence, while the stop codon of JP2 was removed by site-directed mutagenesis. Sequences were confirmed by DNA sequencing. Recombinant JP2 was expressed as N-terminally GST-tagged fusion protein in Escherichia coli BL21 induced by addition of 1.0 mM IPTG at OD 0.6 followed by incubation at 30 °C for 4 h. After cell lysis and centrifugation, the protein in the supernatant was affinity purified on Gluthatione Sepharose 4B prepacked columns (GE Healthcare). The tag was removed by on-column cleavage using PreScission Protease (GE Healthcare) resulting in the elution of untagged JP2. Finally, JP2 was purified by size-exclusion chromatography using a HiLoad 16/60 Superdex 200 column (GE Healthcare) in 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM DTT, 1% Triton X-100.