Histrap hp

Wild-type FLuc was purchased from Promega, or purified as follows. Wild-type and mutant FLuc constructs were transformed into E. coli BL21 (DE3) cells. Cells were grown at 37 °C until OD₆₀₀ reached ~0.4. The temperature was then shifted to 18 °C and cultures were incubated for 1 h before addition of IPTG to a final concentration of 0.25 mM. Proteins were expressed for 16 h at 18 °C. Cells were harvested by centrifugation at 5000g for 30 min and pellets were resuspended in lysis buffer (50 mM NaH₂PO₄ pH 8.0, 300 mM NaCl, 10 mM Imidazole, 1 mg mL⁻¹ lysozyme, PMSF, aprotinin, PepA, leupeptin, DNase I and Complete^TM, EDTA free protease inhibitor cocktail (Roche)). Cells were lysed by sonication, and the lysate was cleared by centrifugation at 66,000g for 30 min. The supernatant was loaded onto a 5 mL Ni-NTA column (HisTrap^TMHP, GE Healthcare Life Sciences) pre-equilibrated with equilibration buffer (50 mM NaH₂PO₄ pH 8.0, 300 mM NaCl, 20 mM imidazole). Protein was eluted in 2 mL fractions using equilibration buffer containing 500 mM imidazole, and purity of the fractions was assessed by 12% SDS-PAGE. Fractions containing pure FLuc were pooled, concentrated, and buffer exchanged into FLuc storage buffer (25 mM Tris-acetate pH 7.8, 1 mM EDTA, 0.2 M ammonium sulfate, 15% glycerol, 30% ethylene glycol, 2 mM TCEP) before snap-freezing in liquid N₂ and storage at −80 °C.

The gene coding for α-HL WT and α-HL M113G were custom synthesized and constructed in a pet 30a(+) plasmid (Genscript, New Jersey) for prokaryotic protein expression. Heptameric α-HL were expressed with E. coli BL21 (DE3) and purified with nickel affinity chromatography. After heat shock transformation with plasmid gene coding for either α-HL WT or α-HL M113G, the cells were grown in LB medium at 37 °C till OD₆₀₀ = 0.7. Isopropyl β-D-thiogalactoside (IPTG) was then added to a final concentration of 1 mM for induction. After shaking overnight at 18 °C, the cells were harvested by centrifugation (2546 × g, 20 min, 4 °C). The pellet was collected and re-suspended in lysis buffer 1 (0.5 M NaCl, 20 mM HEPES, 1% Triton X-100, pH = 8.0), sonicated for 15 min and then centrifuged (18,800 × g, 4 °C, 40 in) to remove intact cells. After syringe filtration, the supernatant was loaded onto a nickel affinity column (HisTrapTM HP, GE Healthcare). After washing the column with buffer A1 (0.5 M NaCl, 20 mM HEPES, 5 mM imidazole pH 8.0), the α-HL heptamers were then eluted with a linear gradient of imidazole to buffer B1 (0.5 M NaCl, 20 mM HEPES, 500 mM imidazole, pH 8.0). Fractions of interests were further characterized and confirmed with SDS-polyacrylamide gel electrophoresis (Supplementary Fig. 3). Only fractions of the α-HL heptamer were collected for subsequent electrophysiology.

Escherichia coli BL21 (DE3) cells transformed with the PET28c expression vector and polyHistidine (pHis) constructs encoding for N-terminally hexa-histidine-tagged TcTR were kindly donated by Dr. Carlos Robello. Expression and purification were performed as described by Arias et al. [31 (link)]. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added at a final concentration of 0.5 mM to overnight cultures of transformed E. coli grown to exponential phase (OD₆₀₀ of 0.6). After 20 h incubation with orbital shaking at 20 °C, cells were harvested by centrifugation (5000× g, 10 min, 4 °C). The bacterial pellet was resuspended in buffer A (20 mM Tris–HCl, pH 7.5, 400 mM NaCl, 10 mM imidazole) and 1 mM FAD cofactor. The cell suspension was lysed using an Ultrasonic homogenizer (JY 92-IN) and debris was removed by centrifugation and filtration. The obtained sample was loaded onto a 1 mL HisTrap^TM HP (GE Healthcare). The column was washed with buffer A and 30 mM imidazole, and the recombinant protein was eluted with 100% buffer B (20 mM Tris–HCl, pH 7.5, 400 mM NaCl, 300 mM imidazole) in 10 column volumes. After analysis by SDS-PAGE, the fractions containing the pure enzyme were pooled. Protein concentration was determined from the absorbance of the flavin prosthetic group at 458 nm (ε₄₅₈= 11.2 mM⁻¹ cm⁻¹) [32 (link)], with a Cary 60 UV-Vis spectrophotometer (Agilent Technologies).

For expression of recombinant Gal, the plasmid pET-gal was transformed into E. coli BL21 (DE3) and cultivated at 37 °C in lysogeny broth (LB) containing 30 μg/mL kanamycin until the OD₆₀₀ of the culture reached 0.6–0.8. Following this step, isopropyl-β-d-thiogalactopyranoside (IPTG) was added to the culture with a final concentration of 0.2 mM and the culture was further grown at 16 °C for 12 h. The cells were harvested by centrifugation at 8000 rpm for 10 min at 4 °C and suspended in a lysis solution (50 mM sodium phosphate buffer, pH 8). The mixture was then disrupted by sonication and the cell debris removed by centrifugation at 13,000 rpm for 15 min. The filtered supernatant was applied to a Ni²⁺-chelating affinity column (His-Trap^TM HP, GE, Madison, WI, USA) which was previously equilibrated using an equilibration buffer (50 mM sodium phosphate buffer, 20 mM imidazole, 300 mM NaCl, pH 8) and subsequently eluted using a linear gradient of 20–250 mM imidazole in equilibration buffer. Enzyme purity was analyzed by SDS-PAGE (GenScript, Nanjing, China). Purified recombinant Gal was buffer-exchanged to 50 mM sodium phosphate buffer (pH 8) via centrifugal ultrafiltration (MW cut off 10 kDa, Millipore, Burlington, MA, USA). The protein concentration was determined using the BCA protein assay kit (Solarbio, Beijing, China) with bovine serum albumin as a standard.

hnRNPA2 LCD-MBP (containing a His tag on the MPB tag) (region R188-Y341, Fig. S6) of hnRNPA2 (UniProt P22626) (Addgene plasmid #98661; http://n2t.net/addgene:98661; RRID: Addgene_98661) was a gift from Prof. N.L. Fawzi (Brown University, Providence, RI, USA).
Precultures were added to LB medium and incubated until they reached an OD600 = 0.6–0.8 at 37 °C, 170 rpm. 1 mM IPTG was added to induce hnRNPA2 LCD-MBP expression. After incubating for 4 h, the cells were harvested by centrifugation at 4 °C, 5000 rpm for 20 min, flash-frozen, and stored at −80 °C.
The cells were thawed and resuspended in lysis buffer: 100 mM KCl, 50 mM HEPES, 0.5 M NaCl, 10 mM Imidazole, pH 7.5 supplemented with 1 mM dithiothreitol, 0.1 mM PMSF, 0.5 mM BA, and 1 tablet Roche complete EDTA-free protease inhibitor per 50 ml on ice. The cells were lysed by sonication (on a Sonics VCX-70 Vibra cell) for 10 min (5 s pulse on, 5 s pulse off; 70% amplification) on ice to avoid heating the sample.
Contaminants were pelleted by centrifugation at 20 000 × g for 1 h at 4 °C. The supernatant was filtered through a 0.45-μm pore filter and loaded onto a Nickel-charged IMAC column (HisTrapTM HP – GE Healthcare) and eluted with a linear imidazole gradient 0–250 mM. The remaining contaminants were removed by gel filtration and the protein was either immediately used or stored for a few days at 4 °C.

The expression of the selected clone was produced in a 1000 mL Erlenmeyer cell culture flask, as described above (expression of scFv section). For medium-scale purification, His₆-tagged scFv fragments were purified by immobilized-metal (Nickel) affinity chromatography (HisTrap^TM HP, GE Healthcare Life Sciences, USA) according to the manufacturer’s instructions in a HPLC system (AKTA^TM purifier). The purified scFv was concentrated using a 10 kDa Amicon apparatus (Millipore, USA), the protein concentration was determined26 (link) and a dot-blot assay of the purified scFv was performed18 (link) to confirm the efficiency of the purification process.