Akta protein purification system

Recombinant serotype 4 polysaccharide was produced in E. coli, as previously described [28] (link). Conjugation to AcrA was carried out using protein glycan coupling technology [25] (link). E. coli cultures were grown for 16 h. These starter cultures were used to inoculate 2 L of SSOB to an OD600 of 0.03 and incubated with shaking at 28 °C. Once OD600 had reached 0.4–0.6, expression of PglB was induced with the addition of 1 mM IPTG. MnCl₂ was also added to a final concentration of 4 mM. After 20 h growth at 28 °C cells were pelleted by centrifugation at 5400g for 30 min at 4 °C. Pellets were resuspended in lysis buffer (50 mM NaH₂PO₄, 0.3 M NaCl and 10 mM imidazole, pH 7.5) with 1 mg/ml lysozyme, and lysed using a FastPrep instrument (MP Biomedicals) with lysing matrix B. Supernatant was treated with 250 units benzonase for 10 min. Insoluble debris was removed by centrifugation at 7800g for 60 min at 4 °C and the supernatant passed through a 0.2 µm filter. The protein/polysaccharide conjugate labeled with a polyhistidine affinity tag was purified using HisTrap columns (GE Healthcare) using an imidazole gradient of 20–300 mM on an AKTA protein purification system (GE Healthcare).

The prepared EAH sepharose 4B was packed to the column of an AKTA protein purification system (GE Healthcare, Beijing, China). The packing flow rate was maintained for three bed volumes after reaching a constant bed height was reached.
After loading the BBMVs of the M. separata, the medium was washed with the binding buffer (0.1 mol/L NaH₂PO₄/Na₂HPO₄, pH 7.4 containing 0.5 mol/L NaCl) until the base line become stable. For competitive elution, periplocoside E was dissolved in the binding buffer. The elution peak was collected by washing the medium with the elution buffer. The affinity medium was then washed with an alternating high pH (0.1 mol/L Tris-HCl, pH 8.0 containing 0.5 mol/L NaCl) and low pH (0.1 mol/L acetic acid/ sodium acetate, pH4.0 containing 0.5 mol/L NaCl) buffer solution to regenerate the medium. This cycle was repeated three times.

Filtered soluble recombinant wild-type RBPMS-A and mutants were purified on an AKTA protein purification system (GE Healthcare;). Recombinant protein was first purified on HisTrap HP column (GE Healthcare) using Buffer A (50 mM Tris Base, 0.5 M KCl, 40 mM imidazole, 10% glycerol, 1 mM DTT, pH 8.5 at 4°C), Buffer A2 (50 mM Tris base, 2 M NaCl, 10% glycerol, 1 mM DTT, pH 8.5 at 4°C), and Buffer B (50 mM Tris base, 0.5 M KCl, 500 mM imidazole, 10% glycerol, 1 mM DTT, pH 8.5 at 4°C). After fraction analysis on SDS-PAGE, the fractions containing recombinant protein were buffer exchanged to Buffer QA (25 mM CAPS, 50 mM KCl, 10% glycerol, pH 10 at 4°C) followed by TEV protease cleavage at a ratio of 1:40 for 2 h at 30°C and stored overnight at 4°C. Cleaved recombinant protein was then purified on a MonoQ 5/50 GL column (GE Healthcare) using Buffer QA (described above + 1 mM DTT) and Buffer QB (25 mM CAPS, 50 mM KCl, 1 M NaCl, 10% glycerol, 1 mM DTT, pH 10 at 4°C). Fractions with pure protein, as analyzed by SDS-PAGE (Supplementary Figure S6), were pooled, quantified on a NanoDrop spectrometer (Thermo Scientific), concentrations determined using predicted extinction coefficients, and stored at −80°C.

Native and recombinant forms of ArtinM were submitted to size exclusion chromatography for molecular weight determination, on a Superdex 75 column (Sigma Aldrich) coupled to an AKTA protein purification system (GE Healthcare, Uppsala, Sweden), which was calibrated by using protein molecular weight standards (Protein Mixture, GE Healthcare). The molecular weight of proteins was determined by partition coefficient (Kav) using this formula: Kav = Ve-Vo/Vt-Vo, where Ve is the elution volume of the samples, Vt is the total volume and Vo is the void volume of the gel bed. High molecular weight blue dextran was used to determine the void volume.

The recombinant plasmids pCold1-S1 and pCold1-S1-PA were transformed into competent E. coli BL21 (DE3) cells (Tiangen Biotech, #CB105) using the heat shock method. The transformed E. coli cells were inoculated on Luria Broth (Amp⁺/LB) Agar plates containing 50 μg/mL ampicillin and cultured at 37°C for 18 h. The single colony containing the target gene was selected and inoculated into Amp⁺/LB broth for culture at 37°C. When the OD_{600 nm} value of the bacterial culture reached 0.6-0.8, the culture was moved to 4°C for 1 h. After adding 0.5 mmol/L isopropyl-β-d-thiogalactoside (IPTG), the bacteria were cultured at 16°C for 24 h to induce the expression of recombinant proteins. The bacterial cells were harvested and re-suspended in PBS (pH 7.4), followed by sonication in an ice bath, the supernatant was collected for SDS-PAGE analysis after centrifugation at 10,000 r/min for 10 min. The expressed proteins were purified using the HisTrap™ HP (GE company, #17524801) on the AKTA protein purification system (GE company, USA), and confirmed by Western blotting. The protein concentration was measured using the BCA protein assay kit (Takara Bio, #T9300A).

The percent monomer of purified antibodies was determined by size exclusion chromatography. 0.1 mg of purified antibody was injected into the AKTA protein purification system (GE Healthcare Life Sciences) and protein fractions were separated using a Superdex 200 10/300 column (GE Healthcare Life Sciences) with 50 mM Tris (pH 7.5) and 150 mM NaCl. The elution profile was exported as Excel file and chromatogram was developed. The protein sizes were determined by comparing the elution profile with the gel filtration standard (Bio-Rad 151–1901) (55 (link)). Any protein peak observed in void fraction was considered as antibody aggregate. The area under the curve was calculated for each peak and a relative percent monomer fraction was determined as described earlier (20 (link)).