Production and Purification of Glycoconjugate Vaccine

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).