Superdex 200 prep grade column

DNA encoding amino acid 38 to the C terminus of CtpA was subcloned into plasmid pET15b between the NdeI and XhoI sites to encode N-terminal His₆-tagged CtpA(ΔN37). Similar plasmids encoding CtpA-S302A, CtpA(ΔC6), CtpA-L426K L430K, or CtpA-L426A L430A were generated by site-directed mutagenesis. For all CtpA proteins, E. coli BL21(DE3) transformants were grown at 37°C to optical density at 600 nm (OD₆₀₀) = 0.6 to 0.7 before being induced with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 16°C overnight. Cells were lysed by passing through a microfluidizer cell disruptor in 10 mM potassium phosphate, pH 8.0, 10 mM imidazole, 0.25 M NaCl. The homogenate was clarified by centrifuging at 27,000 × g, and the supernatant was applied to a HiTrap-Ni column (GE Healthcare) preequilibrated with lysis buffer. Proteins were eluted with a 10 to 300 mM imidazole gradient in 10 mM potassium phosphate, pH 8.0, containing 0.25 M NaCl. Fractions containing His₆-CtpA were collected. The N-terminal His tag was removed using thrombin (0.5 units/mg) by dialyzing against 20 mM Tris, pH 8.0, 150 mM NaCl overnight at 4°C. Untagged CtpA was further purified with HiTrap-Q in 10 mM Tris, pH 8.0, and a 50 to 500 mM NaCl gradient and polished by gel filtration in 10 mM Tris, pH 8.0, and 150 mM NaCl using Superdex 200 prep-grade column (16 × 600 mm, GE Healthcare).

Gel filtration chromatography was carried out by the AKTA-FPLC system (GE Healthcare Bio-Sciences, Piscataway, NJ, USA). In brief, purified protein (10 mg/mL) in Buffer B (20 mM MES and 100 mM NaCl, pH 6.0) was applied to a Superdex 200 prep grade column (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) equilibrated with the same buffer. This column was operated at a flow rate of 0.5 mL/min, and the proteins were detected at 280 nm. The column was calibrated with proteins of known molecular weight: thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B₁₂ (1.35 kDa).

BsAb (EphA10/CD3) and BsAb (His/CD3) were prepared using the Expi293 Expression System (Invitrogen). Plasmid DNA (300 μg) and ExpiFectamin 293 Reagent (800 μL) were mixed with Opti-MEM® I medium (15 mL final volume) and allowed to stand at room temperature for 25 min. The mixed solution was added to 7.5 x 10⁸ Expi293 cells cultured in Expi293 Expression Medium and gently mixed in a shaker incubator at 37°C with a humidified atmosphere of 8% CO2 in air. At 18 hours post-transfection, 1.5 mL of ExpiFectamin 293 Transfection Enhancer 1 and 15 mL of ExpiFectamin 293 Transfection Enhancer 2 were added to each flask. The transfected cells were then incubated under the same conditions in a shaking incubator for one week.
Each BsAb was purified from the cell culture supernatant by immobilized metal affinity chromatography (IMAC) and gel filtration chromatography with a Superdex200 prep grade column (GE Healthcare, Little Chalfont Bucks, UK) equilibrated in phosphate-buffered saline (PBS). SDS-PAGE and western blot analysis were performed to detect and confirm the size and purity of each BsAb. Purified proteins were concentrated in PBS by ultrafiltration using a Centriprep^® 30K or 50K device (Millipore, Billerica, MA, USA), and protein concentrations were estimated using a Coomassie Plus Protein Assay (Thermo Fisher Scientific, Rockford, IL).

BL21(DE3) cells containing GST-Sos1 were collected by centrifugation and lysed by sonication in 25 mM Tris-HCl (pH 8.0), 200 mM NaCl, 2 mM EDTA (pH 8.0), 1 mM DTT, 1 mM PMSF, 1 μg/ml antipain, 1 μg/ml pepstatin, and 1 μg/ml leupeptin. Centrifuge-cleared lysate was applied to Glutathione Sepharose 4B (GE Healthcare) and washed with 50 mM HEPES (pH 7.0), 150 mM NaCl, 10% glycerol and 1 mM DTT. GST was cleaved from protein by PreScission protease treatment overnight at 4°C. Cleaved protein was purified by size exclusion chromatography using a Superdex 200 prepgrade column (GE Healthcare) in 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM βME, and 10% glycerol.

For Sti1 variants and yHsp90 (Hsp82), pET28 vectors carrying the respective genes plus an N-terminal 6 × His-SUMO-tag or a Thrombin cleavable 6xHis-tag were transformed into the E. coli strain BL21 (DE3) Codon Plus. Protein expression was induced at OD₆₀₀ of 0.5 by addition of 1 mM isopropyl-β-D-thiogalactopyranosid (IPTG) overnight at 30 °C. In the case of full-length yHsp70, Pichia pastoris strain KM71H-Ssa1 (aox1::ARG4; arg4; 6xHis-SSA1 gene genomically inserted at AOX1 locus) was used for expression that was induced with 0.5% (v/v) methanol. Proteins were first purified by a 5-ml Hi-Trap column (GE Healthcare). After cleavage of the His₆-SUMO tag or His-tag, respectively, gel filtration chromatography was performed with a Superdex 200 PrepGrade column (GE Healthcare) pre-equilibrated in 40 mM Hepes (pH 7.5), 150 mM KCl, 5 mM MgCl₂. Point mutations of Sti1 were generated using the QuikChange II Site-Directed Mutagenesis Kit (Agilent).