Superdex 200 pg 16 60 column

The supernatant of the transfected cell cultures were collected a fortnight later and spun down at 4,000 rpm, 4°C, for 1 h before 0.2 µm filtration and purified using the ÄKTA pure system (GE Healthcare) using Protein G affinity column for the IgGs as previously described (15 (link)). The 5 ml HiTrap TALON crude affinity column (GE Healthcare) was used for the recombinant FcγIIA using the same settings. The affinity purified proteins were subsequently gel filtrated using the Superdex 200 pg 16/60 column (GE Healthcare) precalibrated with Gel filtration HMW calibration kit (GE Healthcare) and the Gel filtration LMW calibration kit (GE Healthcare) to extract the monomeric fractions as previously described (14 (link), 15 (link)). Pure IgG1 variants and FcγIIA fractions were collected and concentrated using 100 kDa Amicon Pro System (Merck) and 10 kDa Amicon Pro System (Merck) concentrators, respectively. The final concentrations of the proteins were determined by spectrophotometric analyses using nanodrop (Thermo Fisher Scientific) with consideration of calculated protein extinction coefficients of the various IgG1 variants and FcγIIA.

A gene encoding C-terminally His6-tagged GalE (pET-30-lnpD, residues 1–340) was cloned from the genomic DNA of B. longum JCM1217 as previously described^{15 (link)}. The expression plasmid was introduced into E. coli BL21 CodonPlus (DE3)-RIL (Stratagene, La Jolla, CA) for protein expression. The transformant was cultivated at 37 °C in Luria-Bertani medium containing 100 mg/L kanamycin and 20 mg/L chloramphenicol until the absorbance at 600 nm reached 0.6. Protein expression was induced by adding 0.1 mM isopropyl-β-D-thiogalactopyranoside to the medium. The culture was incubated for an additional 20 h at 25 °C. Expressed cells were harvested by centrifugation and suspended in 50 mM Tris-HCl (pH 7.0) and 0.1 mM phenylmethylsulfonyl fluoride. The cells were disrupted via sonication, and the supernatant was purified by sequential column chromatography. Ni-affinity chromatography was conducted using a HisTrap FF crude column (GE Healthcare, Fairfield, CT) with two steps of 20 mM and 250 mM imidazole in 50 mM Tris-HCl (pH 7.0). Gel-filtration chromatography was conducted using a Superdex 200 pg 16/60 column (GE Healthcare) equilibrated with 50 mM Tris-HCl (pH 7.0) and 150 mM NaCl at a flow rate of 1 mL/min. The purified protein concentrations were determined by a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA) with bovine serum albumin as the standard.

The suspension with the harvested cells was defrosted and 90 µl of DNAse was added (∼2 µg ml⁻¹). The cell suspension was sonicated using a Fisherbrand Model 705 Sonic Dismembrator in three 2 min cycles. The disrupted cells were centrifuged (4°C and 14 000 rev min⁻¹ for 1 h). The protein was then filtered and loaded onto a BioLogic DuoFlow FPLC system (Bio-Rad, USA) equilibrated in buffer A (10 mM Tris, 50 mM sodium formate, 10 mM imidazole pH 8.5) and buffer B (10 mM Tris, 50 mM sodium formate, 500 mM imidazole pH 8.5). The protein was purified by metal-affinity chromatography using a nickel-charged column at a flow rate of 1 ml min⁻¹. The protein was eluted with an increasing gradient of imidazole (0%, 10%, 60% and 100% buffer B; DmmarA was eluted in the 10% and 60% gradients). The purified protein from the 60% imidazole gradient was concentrated to ∼5 ml using Amicon Ultra-15 Centrifugal Filter Units (10 kDa cutoff).
5 ml of protein was loaded onto an ÄKTApure FPLC system (Cytiva, Sweden) equilibrated in gel-filtration buffer (10 mM Tris, 50 mM sodium formate pH 8.5). The protein was purified by size-exclusion chromatography using a Superdex 16/60 200 pg column (GE Healthcare, UK). SDS–PAGE was used to check the purity of fractions from affinity chromatography and size-exclusion chromatography (run at 200 V and 400 mA for 40 min).

The suspension with the harvested cells was defrosted and 90 µl of DNAse was added (∼2 µg ml⁻¹). The cell suspension was sonicated using a Fisherbrand Model 705 Sonic Dismembrator in three 2 min cycles. The disrupted cells were centrifuged (4°C and 14 000 rev min⁻¹ for 1 h). The protein was then filtered and loaded onto a BioLogic DuoFlow FPLC system (Bio-Rad, USA) equilibrated in buffer A (10 mM Tris, 50 mM sodium formate, 10 mM imidazole pH 8.5) and buffer B (10 mM Tris, 50 mM sodium formate, 500 mM imidazole pH 8.5). The protein was purified by metal-affinity chromatography using a nickel-charged column at a flow rate of 1 ml min⁻¹. The protein was eluted with an increasing gradient of imidazole (0%, 10%, 60% and 100% buffer B; DmmarA was eluted in the 10% and 60% gradients). The purified protein from the 60% imidazole gradient was concentrated to ∼5 ml using Amicon Ultra-15 Centrifugal Filter Units (10 kDa cutoff).
5 ml of protein was loaded onto an ÄKTApure FPLC system (Cytiva, Sweden) equilibrated in gel-filtration buffer (10 mM Tris, 50 mM sodium formate pH 8.5). The protein was purified by size-exclusion chromatography using a Superdex 16/60 200 pg column (GE Healthcare, UK). SDS–PAGE was used to check the purity of fractions from affinity chromatography and size-exclusion chromatography (run at 200 V and 400 mA for 40 min).