Gel filtration protein standard

The native confirmation of LdG5K was confirmed by size exclusion chromatography using a Superdex 200 H/R 10/30 column fitted onto an AKTA FPLC system (both from GE Healthcare) with a buffer comprising 50 mm Tris‐HCl, pH 7.2 and 0.15 m NaCl running at 0.5 mL·min⁻¹ at room temperature. Gel filtration protein standards (Bio‐Rad) used were as follows: vitamin B12 (1350 Da); horse myoglobin (17 000 Da); chicken ovalbumin (44 000 Da); bovine γ‐globulin (158 000 Da) and bovine thyroglobulin (670 000 Da). The void volume was 8.3 mL. The equation for estimating the molecular mass was derived from plots of V_e/V_o against log MW of the standards.

Analytical gel filtration chromatography was performed on Protein-PAK 200SW column with dimensions of 8.0 mm (i.d.) × 30 cm (glass; P/N WAT011786; Waters Associates) equilibrated with chromatographic buffer containing 10 mM sodium phosphate buffer, pH 7.4. 100 μl of peptide solution corresponding to 0.1 mM concentration was loaded into the equilibrated column and run at a flow rate of 0.5 ml/min on an ǞKTA purifier system (Cytiva). In the presence of the detergent micelles, the column was equilibrated with the chromatographic buffer containing 10 mM n-dodecyl β-D-maltoside (CMC in H₂O: ∼0.17 mM) and the peptides were incubated in the corresponding detergent for 30 min at 4°C prior loading onto the column. Gel filtration protein standards (Bio-Rad) were used for calibration of the column equilibrated with chromatographic buffer.

Antibody monomers, dimers, and high molecular weight (HMW) aggregates were isolated from preparations of IVIg, pAbs and mAbs using a HiPrep 16/60 Sephacryl S-300 HR or a Superdex 200 Increase 10/300 GL column (GE Healthcare). The columns were equilibrated in PBS, pH 7.4, and calibrated using gel filtration protein standards (Bio-Rad). The size of IgG conformers present in SEC fractions was estimated by DLS. SEC fractionated IgGs were used immediately for ELISA or for generating heat-induced antibody aggregates.

20 µg purified protein sample (100 μL of 0.2 mg/mL stock diluted in PBS, pH7.4) was injected onto either a Superdex 200 10/300 GL Tricorn column (GE Healthcare, Little Chalfont, UK) or a TSK Gel G3000SWXL, 7.8 × 300 mm, column (Tosoh Bioscience, Reading, UK) 3 days post-purification and developed respectively with an isocratic gradient of PBS, pH 7.4 at 1 mL/min or 200 mM phosphate, pH 7.0 at 1 mL/min. Signal detection was by absorbance at 280 nm. Gel filtration protein standards (BioRad, Watford, UK) were loaded for molecular weight estimation (chromatograms are shown in Supplementary Figure S1). Since all purified fusion proteins contain disulphide stabilised Fv/scFv, the observed monomer level is independent of protein concentration and unaffected by on-column dilution effects.

Size exclusion chromatography experiments were performed using a Superdex 200 Hiload 16/600 column (GE) with a General Electric AKTA FPLC system. Before loading samples, the size column was equilibrated with 2 column‐volume of buffer (20 mmol L⁻¹ Tris–HCl (pH 8.0), 300 mmol L⁻¹ NaCl). To obtain HisPrgX‐C and HisPrgX‐I used for size exclusion experiments, nickel affinity columns were first bound with HisPrgX (from 700 ml culture), C or I (200 μg) dissolved in DMF was then added to the column and incubated at RT for 20 min. After extensive washing, protein‐peptide complex was eluted using buffer: 20 mmol L⁻¹ Tris‐HCl, pH 8.0, 300 mmol L⁻¹ NaCl, 1 mol L⁻¹ imidazole. Affinity purified HisPrgX, HisPrgX‐C or HisPrgX‐I were loaded to the size exclusion column. The elution was analyzed by monitoring UV absorbance at 280 nmol L⁻¹. The column was calibrated using gel‐filtration protein standards (Bio‐rad). Log₁₀ (Molecular weight) was plotted against Ve/Vo (Ve is the elution volume at maximum A₂₈₀ absorbance for a given sample and Vo is the void volume of the column determined to be 9.58 ml based on the elution of Dextran blue). The molecular weights of eluted complexes were determined by the elution volumes (Ve) and the equation from standard curve.