Pet sumo vector

The EstATII-TM was amplified from a p-GEM cloning vector containing the EstATII ORF [2 (link)] using the forward prime (EstF) 5′-ATG TCC AGG TAC GTT GAT GAG C-3′, reverse primer (EstR) 5′-TCA GCT TAC CGA GTC GGT CT-3′, and DreamTaq Master Mix (Thermo Fisher SCIENTIFIC, Waltham, MA, USA) according to the manufacturer’s instructions. A 945 bp band was observed on an agarose gel (Figure 3a). The amplified ORF was cloned into the pET-SUMO vector (Thermo Fisher SCIENTIFIC, USA). The recombined expression plasmid was transformed into the TOP10 Chemically Competent E. coli cells, as previously stated [39 ]. Both PCR (using ORF’s forward primer and the vector’s reverse primer) and double restriction digestion with spaI and HindIII were performed to verify the correct orientation of the EstATII-TM ORF. The plasmid with the EstATII-TM gene inserted in the correct frame (colony C4) was used for expression in E. coli BL-21 (DE3) (Figure 3b).

For expression in HEK293T cells, cDNA coding for CASK transcript variant 3 (TV3; (Tibbe et al, 2021 (link))) fused to an N-terminal mRFP-tag in pmRFP-N1 was used. For expression in neurons, cDNAs coding for mRFP-CASK-TV5 fusion proteins were inserted into a vector carrying the human synapsin promoter (Repetto et al, 2018 (link); Tibbe et al, 2021 (link)). For the preparation of fusion proteins, the cDNA encoding the CaMK domain was cloned into the pET-SUMO vector coding for a His₆-SUMO tag (Thermo Fisher Scientific). An expression vector for GFP-Mint1 was obtained from C Reissner and M. Missler. HA-tagged Neurexin-1β was from P. Scheiffele via Addgene (58267), HA-tagged, and GFP-tagged Liprin-α2 were from C. Hoogenraad. Mutations were introduced using the Quik-Change II site-directed mutagenesis kit (Agilent), using two complementary, mutagenic oligonucleotides. Constructs were verified by Sanger sequencing.

The EFAU004_01209 PCR purified product was cloned into the pETSumo vector (Thermo Scientific) according to the manufacturer’s instructions. The expression and purification of the recombinant EFAU004_01209 (rEFAU004_01209) under native conditions was performed as described previously^{40 (link)}. The eluted rEFAU004_01209 protein was dialyzed against PBS (phosphate buffered saline).

The codon-optimized DNAs encoding Bcl-xLΔTM (the flexible loop, residues 27–82, and the transmembrane domain, residues, 197–233, are deleted, total 153 residues, named as Bcl-xL thereafter) was ordered from Genewiz (www.genewiz.com). The recombinant Bcl-xL with N-terminal SUMO-fused 6× His tag was generated by the insertion of the PCR products into pETSUMO vector (Invitrogen; www.thermofisher.com) and verified by sequencing. Recombinant proteins were expressed in Escherichia coli (BL21/DE3) strain overnight at 20 °C and proteins were induced by 0.4 mM isopropyl β-d-thiogalactoside. Cells were harvested by centrifugation and the pellets were re-suspended and passed through a cell disruptor (www.avestin.com) three times. After ultracentrifugation at 40,000 rpm for 1 h, the supernatant for His-tagged recombinant proteins were purified through Ni²⁺ affinity column, cleaved by Ulp-1, followed by HiLoad Superdex S-75 26/60 column (GE Healthcare). The purified proteins were dialyzed against stabilization buffer containing 20 mM Tris-HCl (pH 7.0) and 100 mM NaCl and concentrated to 10–15 mg/ml in a Centriprep-30 (Amicon) for subsequent crystallization and biochemical analysis.

The DNA sequences encoding C1B-C2 (residues 100–293), isolated C1B (residues 100–152) or C2 (residues 155–293) of PKCα (M. musculus for C1B-C2 and C1B; R. Norvegicus for C2) were amplified by PCR using the cDNA clone of PKCα (Open Biosystems) as a template and cloned into the pET-SUMO vector (Invitrogen). Isolated C1B, C2, and C1B-C2 were expressed and purified as described previously (Morales et al., 2011 (link); Stewart et al., 2011 (link); Cole et al., 2019 (link)). [U-¹⁵N, 75%-²H]-enriched C1B-C2 and [U-¹⁵N]- or [U-¹³C, ¹⁵N]-enriched C1B were used for the NMR experiments.