Amicon ultra concentrator

E. coli cells, which expressed the recombinant proteins, were disrupted by sonication in a lysis buffer composed of 50 mM potassium phosphate buffer (pH 7.4), 500 mM KCl, 5 mM MgCl₂, 0.1 mM EDTA, 0.1 mM DTT, 20% (v/v) glycerol, 1.0% (w/v) CHAPS, 5 mM ATP, and 25 mM imidazole. Following centrifugation at 10,000 g for 30 min at 4°C, the supernatant was applied to a TALON spin column (Takara Bio) that had been equilibrated with the lysis buffer. The proteins bound to the column were washed twice with 600 μl of the lysis buffer and then eluted twice with 200 μl of an elution buffer. This elution buffer was composed of 50 mM potassium phosphate buffer (pH 7.4), 500 mM KCl, 0.1 mM EDTA, 0.1 mM DTT, 20% (v/v) glycerol, 1.0% (w/v) CHAPS, and 500 mM imidazole. The eluent was then desalted using buffer A (50 mM potassium phosphate buffer (pH 7.4) and 20% (v/v) glycerol) with a 10 kDa cut-off Amicon Ultra concentrator (Merck Millipore, Burlington, MA, USA). Protein purification was confirmed by SDS-PAGE (Supplementary Figure S3A). The purified proteins were stored at −80°C. The protein concentration was estimated using the Pierce BCA Protein Assay Kit, following the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA).

The recombinant fusion protein containing the ectodomain of human ActRIIB fused to the Fc domain of human IgG1 (sActRIIB-Fc) was produced in-house as described previously [12 (link), 17 (link)]. For the final production of the chimeric protein, CHO cells were transfected with the expression construct via lipofection (Fugene 6; Roche) and selected with puromycin (Sigma-Aldrich). During selection, cells were grown in DMEM supplemented with 2 mM L-glutamine, 100 μl/ml streptomycin, 100 IU/ml penicillin, and 10% fetal calf serum. For large-scale expression, cells were adapted to CD OptiCHO medium (Gibco) supplemented with 2 mM L-glutamine and grown in suspension in an orbital shaker. Cell culture supernatants were clarified by filtration through 0.22 μm membrane (Steritop, Millipore). 1 M NaCl, 5 mM imidazole, and Protease Inhibitor Cocktail (Thermo Scientific) were added into the clarified supernatants, and the solution was pumped through a HisTrap FF Crude column (GE Healthcare Life Sciences, Uppsala, Sweden) at 4°C. Protein was eluted with increasing imidazole concentrations, dialyzed against phosphate-buffered saline (PBS), and finally concentrated with Amicon Ultra concentrator (30 000 MWCO, Millipore).

Full-length cDNA of MSLN was inserted into the baculovirus transfer vector pAcGP67A (RRID:Addgene_41812) of BD BaculoGold (BD Biosciences) in frame with the hexahistidine tag at the C-terminus (Supplementary Table S1). All mutations were made by PCR using the QuikChange mutagenesis kit (Agilent Technologies, Inc.). The plasmid was cotransfected with linearized viral DNA into approximately 2 million Sf9 cells and the culture was gradually amplified to 10 L of cultured insect cells for secretory expression of MSLN. Culture media were collected and concentrated in a diafiltration device (Millipore) against a diafiltration solution containing 25 mmol/L Tris, pH 7.5, 300 mmol/L NaCl, and 10% glycerol. The sample was then mixed with Ni-NTA resin (Qiagen) preequilibrated with the same buffer supplemented with 10 mmol/L imidazole. After washing with the diafiltration buffer supplemented with 50 mmol/L imidazole, bound MSLN was eluted in the presence of 100 mmol/L imidazole. Fractions containing MSLN were pooled and concentrated. MSLN was further purified by size-exclusion chromatography (SEC) using a Superdex 75 column equilibrated with 20 mmol/L Tris, pH 8.0, 100 mmol/L NaCl. Fractions were pooled, concentrated to 16 mg/mL using an Amicon Ultra concentrator (Millipore) with MWCO 30 kDa and stored at −80°C until use.

Cell pellets were resuspended in Ni-NTA buffer and lysed using a microfluidizer (Microfluidics). Cell lysate was cleared by centrifugation at 18000 rpm, 4°C, for 40 min. Supernatant was filtered using a 0.45 μm syringe filter (Millipore) and applied on a 5 mL Ni-NTA column (Macherey Nagel), equilibrated with Ni-NTA buffer. Protein was eluted stepwise using increasing imidazole concentrations; eluted fractions were analyzed by SDS-PAGE. Fractions containing the target protein were pooled and concentrated using an Amicon Ultra concentrator with 30 kDa cut-off (Millipore). Concentrated protein samples were further purified by size exclusion chromatography, on a Superdex 200 pg column (GE Healthcare), equilibrated with SEC buffer (20 mM Tris-HCl, 300 mM NaCl, pH 8.0). All purification steps were performed at a temperature of 4°C. Finally, protein purity was assessed by SDS-PAGE; protein solutions were stored at 4°C.