Cloning, expression, and purification of recombinant proteins

The genes encoding for designed molecules were synthesized as DNA strings by GeneArt (Thermo Fisher Scientific) optimizing the codon usage for expression in the E. coli and adding at the gene extremities the appropriate linker for ligation independent cloning. In order to obtain recombinant N- or C- terminally His-tagged proteins, the genes were cloned into pET15b+TEV and pET21b+ (Merck-Sigma) PCR-amplified vectors using the Infusion cloning kit (Takara) following manufacturer instructions. Protein expression was performed using E. coli BL21(DE3) strain (New England Biolabs). The cells were grown in 500 mL of HTMC media (Glycerol 15 g/L; Yeast Extract 30 g/L; MgSO₄ x7H₂O 0.5 g/L; KH₂PO₄ 5 g/L; K₂HPO₄ 20 g/L; KOH 1 M to pH final 7.35±0.1), under shaking (160 rpm) at 37°C until reaching an optical density OD_600_nm of 0.8 followed by induction with 1mM IPTG for 3h at 37°C. Soluble proteins were extracted by sonication for 10 minutes in 20 mM Tris, 150 mM NaCl and EDTA-free protease inhibitors at pH 8 alternating cycles of 30 s pulse and 30 s stop. The first protein purification step was performed with immobilized metal affinity chromatography (IMAC) using Ni-NTA agarose resin (Thermo Fisher Scientific) and an elution buffer containing phosphate buffered saline (PBS) with 350mM of imidazole. Fractions containing the target protein were applied to a size exclusion chromatography (SEC) column (Superdex 200 10/300, GE Healthcare) equilibrated in PBS buffer, with a flow rate of 0.5 mL/min; the NP proteins were collected in the void volume due to their large sizes. SDS-PAGE analysis was performed to check protein purity and the concentration was determined by UV-Vis absorbance at 280_nm (Nanodrop device).

The electron microscopy analysis was performed loading 5 μl of sample concentrated 20 ng/μL onto a glow discharged copper 300-square mesh grid for 30 s. Blotted the excess, the grid was negatively stained using NanoW for 30 seconds. The samples were analysed using a Tecnai G2 spirit and the images were acquired using a Tvips TemCam-F216 (EM-Menu software).

5 μg of proteins were spotted on a nitrocellulose membrane and let adsorb for 10 minutes. The membrane was then blocked with 3% milk in PBS and 0.1% Tween detergent. The binding with primary antibody 4B3 [56] (diluted 1:1000 in 3% milk) was followed for 1h at room temperature (18–26°C) with gentle shaking, then the membrane was washed three times with 10mL of PBS and 0.1% Tween for 5 minutes each. The secondary antibody conjugated with horseradish peroxidase (HRP) was then added (diluted 1:1000) in 3% milk to the membrane for 1h. Three additional wash steps were performed, as above, before adding the chromogenic substrate 4-chloro-1-naphthol in order to acquire the signal using GelDoc XR+ imaging system.

The capability of human mAb 4B3 [56] to recognize the fHbp1.1 β-barrel nanoparticles was assessed by SPR analysis using the Single Cycle Kinetics method [64]. mAb was diluted to a concentration of 5 μg/mL with running buffer HBS-EP+ (0.01 M HEPES, 0.15 M NaCl, 0.003 M EDTA and 0.05% v/v Surfactant P20) and captured on the surface of a CM5 sensor chip coated with a secondary anti-human IgG Fc. Increasing concentrations (1.25 nM, 2.5 nM, 5 nM, 10 nM, 20 nM) of each analyte were injected for 60 s on the surface of the sensor chip. After the last injection, dissociation of the protein was followed for 1500 s. After each cycle, the sensor chip was regenerated using 3 M MgCl₂. The sensorgram, a plot of response (measured in Resonance Units [RU]) against time (measured in seconds [s]), was used to monitor the interaction. The response is directly proportional to the concentration of biomolecules on the surface. The sensorgrams resulted from the blank subtraction, based on the captured mAb but with injections of buffer instead of samples. Capture adjustment was applied to correct sample responses for variations in the levels of captured mAb between cycles by dividing the sample response with the response for captured ligand. Adjusted response levels are expressed as sample response divided by capture level.