Hiload 26 600 superdex 200 pg column

For expression of IL-12α^C96S,His, ExpiCHO cells (Thermo Fisher Scientific) were cotransfected with IL-12α^C96S,His and IL12β^C199S in a DNA ratio of 1:1 according to the manufacturer’s protocol (high titer). After protein expression for 7 days, the medium was centrifuged (at 5000g for 30 min at 4°C) and applied to a HisTrap HP column (Cytiva). A guanidinium chloride gradient (final 2.5 M GdmCl) was used to separate IL-12β^C199S from IL-12α^C96S,His; subsequent washing with PBS was applied to refold IL-12α^C96S,His on the column. Elution was performed in PBS supplemented with 500 mM imidazole, and the His-tag was optionally cleaved by the addition of TEV protease [TEV: IL-12α^C96S 1:10 (w/w)] overnight at 4°C and removed by a second HisTrap HP column in PBS. Final purification was performed using a HiLoad 26/600 Superdex 200 pg column (Cytiva) in PBS. EBI3 was cotransfected with IL27α^L162C,His into Expi293 (Thermo Fisher Scientific) cells with a DNA ratio of 1:1 according to the manufacturer’s protocol. After 2 days, the medium was centrifuged and applied to a HisTrap column. To separate the complex, a guanidinium chloride wash gradient (final 6 M GdmCl) was used to elute EBI3 from the column, which was further purified by a HiPrep 16/60 Sephacryl S-200 HR in PBS supplemented with 3 M GdmCl. EBI3 containing fractions were pooled, concentrated, and dialyzed against PBS for refolding.

The wild-type and its variants of SpeMreB3 were purified by fusion with a 6 × histidine tag at the N-terminus as follows. Cell pellets harvested from 1-L cultures were resuspended in 20–40 mL buffer A (50 mM Tris–HCl pH 8.0 at 25°C, 300 mM NaCl and 50 mM imidazole-HCl pH 8.0 at 25°C) and sonicated with a probe sonicator (Nissei, Ultrasonic Homogenizer). The cell lysate was then centrifuged (100 000×g at 4°C for 30 min). The supernatant was loaded onto a HisTrap HP column (Cytiva, Wauwatosa, WI, USA), washed with 10 column volumes of buffer A, and eluted with 13 mL of arranged buffer A containing 230 mM imidazole-HCl pH 8.0 at 25°C. The eluted SpeMreBs were further purified using a HiLoad 26/600 Superdex 200 pg column (Cytiva) at 4°C equilibrated with buffer B (20 mM Tris–HCl pH 8.0 at 25°C and 300 mM NaCl). For SpeMreB5 and its variants, the centrifugation strength was decreased to 12 000×g. For purification of SpeMreB3 for the crystallization experiments, buffer C (10 mM Tris–HCl pH 8.0 at 25°C and 150 mM NaCl) was used for gel filtration with a HiLoad 26/600 Superdex 200 pg column instead of buffer B. Protein concentrations were determined from the absorbance at 280 nm measured using NanoDrop One (Thermo Fisher Scientific) with the following absorption coefficients: 0.474 (mg mL⁻¹)⁻¹ cm⁻¹ for SpeMreB3 and its variants and 0.578 (mg mL⁻¹)⁻¹ cm⁻¹ for SpeMreB5 and its variants.

All the preparation methods are described in detail in the Supplementary material. The DNA manipulations were carried out using standard subcloning techniques, and plasmids were propagated in E. coli DH5α^{45 (link),46 (link)}. The (–KTS) isoform of the WT1 gene was obtained by reverse transcription of total RNA isolated from A549 cells using RNeasy mini kit (Qiagene). The E. coli BL21 (DE3) CodonPlus RIL strain was transformed by wild type or each mutant WT1 plasmid by using heat-shock. The isolation of WT1 proteins from the bacterial pellet was achieved by using buffers with increasing concentrations of sodium chloride. The prepared sample was subjected to fast liquid protein chromatography (FPLC, ÄKTA pure protein purification system with Unicorn software) to separate the proteins according their ion charge (HiPrep SP FF 16/10 by Cytiva, strong cation exchange chromatography column). The achieved protein was then separated using high-resolution preparative gel filtration chromatography (HiLoad 26/600 Superdex 200 pg column by Cytiva). The concentration of purified WT1 proteins was calculated and before the MST experiment the samples were concentrated (see Supplementary material for details). The sample buffer was exchanged for the MST binding assay buffer and the final assay concentration was calculated.

All LI-cadherin constructs were expressed as previously described^{9 (link)}. Briefly, Expi293F cells (Thermo Fisher Scientific) were transfected with the pcDNA3.4 vector encoding LI-cadherin sequence and C-terminal Myc-tag, NSAVD sequence, and 6xHis-tag , following the manufacturer’s protocol. Cells were cultured at 37 °C and 8.0% CO₂ for three days after transfection. The supernatant was collected by centrifuging the cell culture for 15 min at 1500 rpm and was dialyzed against a solution of 20 mM Tris–HCl (pH 8.0), 300 mM NaCl, and 3 mM CaCl₂. Proteins were purified by immobilized metal affinity chromatography using Ni–NTA Agarose (Qiagen), followed by size exclusion chromatography using the HiLoad 26/600 Superdex 200 pg column (Cytiva) at 4 °C equilibrated in buffer A (10 mM HEPES–NaOH (pH 7.5), 150 mM NaCl, and 3 mM CaCl₂). Unless otherwise indicated, samples were dialyzed in buffer A before analysis, and the filtered dialysis buffer was used for assays.

Full-length human TPO ORF with a C-terminal 6× His tag, followed by a single stop codon sequence, was synthesized and cloned into pXC17.4 (Lonza Inc., Basel, Switzerland) with HindIII/EcoRI sites, expressed, and purified using the GS Xceed™ Gene Expression System (Lonza). The TPO ORF with a C-terminal 6× His tag in the pXC17.4 vector was transfected into CHOK1SV GS-KO cells by electroporation. For protein purification, TPO-expressing cells were cultured in CD-CHO medium (Thermo Fisher Scientific) for 5 days. The supernatant was collected and loaded onto a HisTrap excel column (Cytiva) equilibrated in 20 mM phosphate (pH 7.4) and 300 mM NaCl buffer. The bound protein was eluted with a phosphate buffer containing 300 mM imidazole. Fractions containing rhTPO-His, as verified by SDS-PAGE, were pooled and loaded onto a 10 mL HiLoad 26/600 Superdex 200 pg column (Cytiva) equilibrated in 2 × PBS. Fractions containing rhTPO-His were added to glycerol at a final concentration of 50% (v/v) and stored at −80 °C until use.

The fusion proteins (EGFP-LecA and LecA-EGFP) were produced in E. coli NiCo21 (DE3) (New England Biolabs GmbH, Frankfurt am Main, Germany) after the transformation of the plasmids pET-28a(+)-EGFP-LecA and pET-28a(+)-LecA-EGFP, respectively. The ÄKTA prime plus chromatography system (GE Healthcare, Leverkusen, Germany) was used for the first round of protein purification. A HisTrap FF Crude column (Cytiva, Freiburg im Breisgau, Germany), which uses an Ni-NTA agarose stationary phase, was utilized in this system. After purification, the fusion protein samples were transferred into a SnakeSkin dialysis tube (Life Technologies GmbH, Darmstadt, Germany) with a rated molecule weight cut-off of 10 kDa and dialyzed overnight with ice-cold DPBS^-/-. The next day, the dialysis buffer was renewed, and samples were continually dialyzed for another 5 h. Then, the fusion protein samples were collected and prepared for size exclusion chromatography (SEC) as a second round of purification. Gel filtration chromatography with DPBS^-/- as a buffer was used as a polishing step for fusion protein purification. Fractions eluted from the affinity column containing EGFP-LecA and LecA-EGFP were pooled together and additionally purified on a HiLoad 26/600 Superdex 200 pg column (Cytiva, Freiburg im Breisgau, Germany) using an ÄKTA avant system (Cytiva, Freiburg im Breisgau, Germany).