Akta chromatography system

Microscale thermophoresis (MST) assay was performed using a NanoTemper Monolith^TM NT.115 instrument (Munich, Germany). His6-OSK1, His6-OSK1^S53D, and His6-TF-SUMO-OsCOI1b were further purified with a prepacked Superdex™ 200 10/300 GL Tricorn™ high-performance SEC column using AKTA chromatography system (GE Healthcare). The proteins were dialyzed with 1× PBS buffer three times at 4 °C. His6-TF-SUMO-OsCOI1b (2 μM, 100 μl) was incubated with 6 μl of the fluorescence dye RED-NHS 2nd generation for 30 m in 25 °C. Labeled His6-TF-SUMO-OsCOI1b (10 μl) was individually mixed with an equal volume of serially-diluted His6-OSK1 or His6-OSK1^S53D in 1× PBS buffer supplied with 0.05% Tween 20. After incubation at 25 °C for 10 m, the mixtures were loaded onto standard-treated silica capillaries (NanoTemper) and fluorescence was measured on 20% LED power and 20% IR-laser power.

As described previously [20 (link),33 (link),34 (link)], BNCs were overexpressed in Saccharomyces cerevisiae AH22R⁻ cells that carried the ZZ-BNC expression plasmid, pGLD-ZZ50. Next, as described previously [20 (link),35 (link)], BNCs were extracted by disrupting the cells with glass beads; then, BNCs were purified on an AKTA chromatography system (GE Healthcare). Next, BNCs were biotinylated with the EZ-Link Sulfo-N-hydroxysuccinimide-biotin kit (Pierce), according to the manufacturer’s protocol. For binding assays, biotinylated BNCs were labeled with Cy3-dye (GE Healthcare) with N-hydroxysuccinimide chemistry, as described previously [36 (link)]. Finally, the drug-containing, biotinylated BNC-LP complex was prepared by conjugating the biotinylated BNC with drug-containing liposomes at a weight ratio of 1:35, as described previously [17 (link)].

A cDNA library was prepared in a previously published study from peripheral mononuclear cells isolated from the blood of a healthy volunteer [12 (link)]. The extracellular region of CLEC-2 (351–890, NM_016509) was amplified by PCR with BamH1 and EcoRV sites at the 5′ and 3′ ends, respectively, using the following primers:
The CLEC-2 sequence was ligated to pFUSEN and amplified, as conducted for the construction of the podoplanin-CSII vector. The DNA sequence for the CLEC-2 region of the vector was confirmed by DNA sequencing. For protein expression, the plasmid was transfected into HEK293 cells, using X-tremeDNA transfection reagent (Sigma), with a DNA to reagent ratio of 2 μg:3 μL. The following day, the culture medium was replaced with Defined K-SFM (Thermo Fisher Scientific 10785) or serum-free MEM-α medium (Nacalai 21444–05) and harvested 2 days later. The pooled medium was loaded onto a HiTrap Protein A HP Column connected to an AKTA chromatography system (GE Healthcare, Chicago, IL, USA). Fc-fusion proteins were eluted with 0.58% acetic acid and immediately neutralized with sodium borate buffer (pH 8.5). The solvent was then replaced with PBS and concentrated by ultrafiltration (Amicon-Ultra-4; Millipore, Bedford, MA). Approximately 300 μg of pure fusion protein was obtained from 1 L of conditioned medium.

All purification procedures were performed using the AKTA chromatography system (GE Healthcare) in a cold room (4 °C); all buffers used contained 0.1% DDM and were adjusted to pH 7.6. Crude extract was applied to a DEAE column (GE Healthcare, DEAE Sepharose Fast Flow) previously equilibrated with buffer A (Tris-HCl, 10 mM, pH 7.6), glycerol (10%) and DDM (0.1%, w/v). The column was eluted with a linear gradient (0 to 500 mM) of sodium chloride supplemented in buffer A, and the QFR was present in the fraction corresponding to sodium chloride (180 to 200 mM) of the eluted buffer. The QFR fraction was subjected to dialysis to decrease the ionic strength in buffer A. This fraction was then loaded onto a Q-sepharose column (GE Healthcare, Q Sepharose Fast Flow) and the protein faction was again eluted with NaCl (~200 mM). As there were some minor contaminations in this step, we performed a final size-exclusion column chromatography (GE Healthcare, Superdex 200 10/300 GL, equilibrated with 20 mM Tris buffer, pH 7.6, containing 10% glycerol and 0.1% DDM) to remove these contaminants. The protein fraction was concentrated in an ultrafiltration cell (Amicon) with molecular-weight cutoff 100 kDa. The purity of the fraction was examined with SDS-PAGE and the UV-visible spectrum (Fig. S3a).

The absolute molar mass of Vfat was determined by multi angle light scattering (MALS). The target protein was loaded onto a Superdex 200 HR 10/30 gel-filtration column (GE Healthcare) that had been pre-equilibrated in a buffer containing 20 mM Tris-HCl pH 8.0 and 150 mM NaCl. The AKTA chromatography system (GE Healthcare) was coupled to a MALS detector (miniDAWM TREOS) and a refractive index detector (Optilab DSP) (Wyatt Technology).