V2.24 fHbp was amplified from N. meningitidis OX99.32412 and SNPs introduced by PCR, then ligated into pET21b using Quick-Stick Ligase (Bioline). Versions of fHbp were ligated into pET28a-His-MBP-TEV (in frame with sequence encoding a histidine tag and the Escherichia coli maltose-binding protein (MBP) with a C-terminal TEV cleavage site) linearised with XhoI, and constructs confirmed by sequencing.
v2.24 fHbps were expressed in E. coli B834 during growth at 22°C for 24 hrs with 1 mM IPTG (final concentration). Bacteria were harvested and resuspended in Buffer A (50 mM Na-phosphate pH 8.0, 300 mM NaCl, 30 mM imidazole) and the fHbp purified by Nickel affinity chromatography (Chelating Sepharose Fast Flow; GE Healthcare). Columns were washed with Buffer A, then with 80:20 Buffer A:Buffer B (50 mM Na-phosphate pH 8.0, 300 mM NaCl, 300 mM Imidazole), and proteins eluted in 40:60 Buffer A:Buffer B. Proteins were dialysed overnight at 4°C into PBS, 1mM DTT pH 8.0 with TEV protease prior to Nickel affinity chromatography to remove the HIS-GST-TEV. fHbp was eluted from Sepharose columns with Buffer B after washing with buffer C (50 mM Na-phosphate pH 6.0, 500 mM NaCl, 30 mM Imidazole), and dialysed overnight at 4°C into Tris pH 8.0. fHbp v1.1I311A expression and purification was performed as described previously [83 (link)].
v2.24 fHbps were expressed in E. coli B834 during growth at 22°C for 24 hrs with 1 mM IPTG (final concentration). Bacteria were harvested and resuspended in Buffer A (50 mM Na-phosphate pH 8.0, 300 mM NaCl, 30 mM imidazole) and the fHbp purified by Nickel affinity chromatography (Chelating Sepharose Fast Flow; GE Healthcare). Columns were washed with Buffer A, then with 80:20 Buffer A:Buffer B (50 mM Na-phosphate pH 8.0, 300 mM NaCl, 300 mM Imidazole), and proteins eluted in 40:60 Buffer A:Buffer B. Proteins were dialysed overnight at 4°C into PBS, 1mM DTT pH 8.0 with TEV protease prior to Nickel affinity chromatography to remove the HIS-GST-TEV. fHbp was eluted from Sepharose columns with Buffer B after washing with buffer C (50 mM Na-phosphate pH 6.0, 500 mM NaCl, 30 mM Imidazole), and dialysed overnight at 4°C into Tris pH 8.0. fHbp v1.1I311A expression and purification was performed as described previously [83 (link)].
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