Resourceq

Bead-bound His-tagged RasGAP²³² was eluted in a stepwise gradient of 20 mM, 40 mM, 100 mM, 250 mM, and 500 mM imidazole in lysis buffer. Fractions containing RasGAP²³² were pooled and mixed with hexahistidine-tagged Tobacco Etch Virus (TEV), and dialyzed overnight against lysis buffer to proteolyze the hexahistidine tag and remove imidazole. The solution was then added back to nickel beads and rocked for 1 h at 4 °C to remove TEV and uncleaved protein. The flowthrough containing tagless RasGAP²³² was concentrated and buffer exchanged into 20 mM Tris pH 8 to reduce NaCl concentration to 50 mM. Anion-exchange chromatography was then performed using either MonoQ 5/50 GL (Cytiva) or ResourceQ (1 ml, Cytiva) columns and buffers A and B which were 20 mM Tris pH 8 and 20 mM Tris pH 8, 1 M NaCl, respectively. Next, size-exclusion chromatography (SEC) was performed using HiLoad 16/600 Superdex 75 prep grade (Cytiva) with 20 mM Tris pH 8, 250 mM NaCl buffer.

For preparation of the Bac1 mutants, E. coli Rosetta2 (DE3) was transformed with each of the expression plasmids. Each transformant was cultured overnight at 37°C in 2 L of LB medium supplemented with 150 μg/ml of ampicillin. Overexpression was induced by the Overnight Express Autoinduction System (Novagen-Merck). The cells were then harvested, resuspended in 20 mM Tris, pH 7.5, 1 mM EDTA, and disrupted by sonication. The cell lysates were centrifuged at 60,000 x g for 20 minutes. The resulting supernatant was individually heat-treated at 75°C for 20 min and then centrifuged again at 60,000 x g for 20 min. To purify the protein, the supernatant was filtered and then subjected to HiTrapQ (Cytiva) column chromatography. The adsorption buffer was 20 mM Tris, pH 7.5, 1 mM EDTA, and the elution buffer was 20 mM Tris, pH 7.5, 1 mM EDTA, 1 M NaCl. The fraction containing the Bac1 mutant was collected and dialyzed overnight against 20 mM Tris, pH 8.8, 1 mM EDTA, followed by ResourceQ (Cytiva) column chromatography. The adsorption buffer was 20 mM Tris, pH 8.8, 1 mM EDTA, and the elution buffer was 20 mM Tris, pH 8.8, 1 mM EDTA, 1 M NaCl. The fractions that were homogeneous judging from the results of Coomassie blue staining after SDS-polyacrylamide gel electrophoresis were used for subsequent analysis.

Bead-bound His-tagged H-Ras^1-167 was eluted in a stepwise gradient of 20 mM, 40 mM, 100 mM, 250 mM, and 500 mM imidazole in lysis buffer. Fractions containing H-Ras^1-167 were pooled and mixed with hexahistidine-tagged TEV and dialyzed overnight against lysis buffer to proteolyze the hexahistidine tag and remove imidazole. The solution was then added back to nickel beads and rocked for 1 h at 4 °C to remove TEV and uncleaved protein. The flowthrough containing tagless H-Ras^1-167 was concentrated and buffer exchanged into 20 mM Tris pH 8 to reduce NaCl concentration to 50 mM. Anion-exchange chromatography was then performed using either MonoQ 5/50 GL (Cytiva) or ResourceQ (1 ml, Cytiva) columns and buffers A and B which were 20 mM Tris pH 8 and 20 mM Tris pH 8, 1 M NaCl, respectively. Next, SEC was performed using HiLoad 16/600 Superdex 75 prep grade (Cytiva) with 20 mM Tris pH 8, 150 mM NaCl buffer.

The mouse IgG2a (mIgG2a) antibody (BALB/c IgG2a,κ Purified Mouse Anti-Mouse I-A[b], Clone AF6-120.1, BD Pharmingen, Catalog No.: 553549. Lot. 5274525), was Azide-activated using the GlyCLICK Azide activation kit (Genovis L1-AZ1-025) according to the manufacturer’s instructions. In brief, 300 µg mIgG2a was concentrated to 1 mg/mL TBS buffer. The antibody was de-glycosylated by head-over-head rotation for 30 min at RT in the GlyCLICK column. The de-glycosylated mIgG2a was separated from the column and incubated with Gal-Az O/N at 30 °C. Azide-activated mIgG2a was concentrated and washed with 6 mL TBS buffer in 100 kDa molecular weight cutoff (MWCO) Amicon (Merck Millipore, cat: UFC210024). The Azide-activated antibody at a concentration of 0.8 mg/mL was conjugated with 7x excess of DBCO-S1-handle with an extension carrying P3 (Table S1) docking site (Metabion Inc.) by incubating O/N at RT and protected from light. To remove unreacted IgG as well as DNA, mIgG2a-DNA-purification was performed on an ÄKTA pure system (Cytiva) using 1 mL RESOURCE Q (Cytiva, cat: 17-1177-01) column. Appropriate fractions were concentrated and buffer-exchanged into PBS via 100 kDa MWCO centrifugal filter. The mIgG2a-DNA conjugate was stored at 4 °C at a concentration of 3.3 μM.

Recombinant proteins were produced using E. coli strain Rosetta2 (DE3) pLysS grown in 2× TY media at 37°C to an A₆₀₀ of 0.4–0.6 followed by induction with 300 μM IPTG and overnight incubation at 18°C.
For in vitro UFMylation assays, in vitro pulldowns, and in vitro protein–protein microscopy binding assays, pelleted cells were resuspended in lysis buffer (100 mM HEPES pH 7.5, 300 mM NaCl) containing protease inhibitors (Complete™, Roche) and sonicated. The clarified lysate was first purified by affinity, by using HisTrap FF (GE HealthCare) columns. The proteins were eluted with lysis buffer containing 500 mM imidazole. The eluted fraction was buffer exchanged to 10 mM HEPES pH 7.5, 100 mM NaCl and loaded either on Cation Exchange (ResourceS, Cytiva) or Anion Exchange (ResourceQ, Cytiva) chromatography columns. The proteins were eluted from 5 to 55% of Ion exchange buffer B (10 mM HEPES pH 7.5, 1 M NaCl by NaCl) gradient in 20 CV. Finally, the proteins were separated by size‐exclusion chromatography with HiLoad^® 16/600 Superdex^® 200 pg or HiLoad^® 16/600 Superdex^® 75 pg, which were previously equilibrated in 50 mM HEPES pH 7.5, 150 mM NaCl.
The proteins were concentrated using Vivaspin concentrators (3,000, 5,000, 10,000, or 30,000 MWCO). Protein concentration was calculated from the UV absorption at 280 nm by DS‐11 FX+ Spectrophotometer (DeNovix).

Enzymes: Ulp1-protease (recombinant, purified in-house), recombinant 4E-BP1 (Sino Biological, 10022-H07E), BamHI-HF (R3136, NEB UK), BsaI-HF (R3535, NEB UK), NotI-HF (R3189S, NEB UK), HindIII-HF (R3104S, NEB UK), NcoI-HF (R3193, NEB UK), NsiI-HF (R3127, NEB UK). Antibodies: eIF4A1 (ab31217, Abcam UK), GFP (ab13970, Abcam UK), vinculin (ab129002, Abcam UK). Kits: HiScribe™ T7 ARCA mRNA Kit (NEB E2065S), HiScribe™ T7 mRNA Kit with CleanCap® Reagent AG (NEB E2080S), Rabbit Reticulocyte Lysate (Promega L4151), TMT 16plex reagent kit (A44522 Thermo Scientific). RNAs: all RNAs were purchased from IBA life sciences or IDT, see Supplementary Table S8. Columns for FPLC: HisTrap 5 ml (17524802, Cytiva), ResourceQ 6 ml (17117901, Cytiva), Heparin 5 ml (17040701, Cytiva), Superdex S200 16/60 (28989335, Cytiva), Superdex S200 increase 3.2/300 (28990946, Cytiva). eIF4A-inhibitors: silvestrol (Generon UK), hippuristanol (gift from John Le Quesne). SILAC amino acids: Lys-¹²C₆¹⁴N₂ (Lys0), Arg-¹²C₆¹⁴N₄ (Arg0), Lys-¹³C₆¹⁵N₂ (Lys8), Arg-¹³C₆¹⁵N₄ (Arg10) all purchased from Cambridge Isotope Laboratories (#ULM-8766, #ULM-8347, #CNLM-291-H, #CNLM-539-H)