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  • br Isothermal titration calorimetry ITC A nity constants and

    2019-10-16


    Isothermal titration calorimetry (ITC). Affinity constants and binding stoichiometry between native/conjugate proteins and mAbs were determined by isothermal titration calorimetry using a VP-ITC MicroCalorimeter (GE Healthcare, Uppsala, Sweden) provided with a ThermoVac accessory for thermostatting and degassing samples. All the samples were dissolved in PBS pH 7.4. The sample Fosfomycin calcium were filled with a mAb solution that was titrated with protein solution loaded in the injector syringe. SpA and SpG concentrations ranged from 10 to 20 μM; mAbs concentration was 1–2 μM. The first injection was of 1 μl, and all the subsequent ones were of 10 μl every 240 s. The injection duration was 20 s and the stirring speed was set at 307 rpm. The experiments were conducted at 30 °C with a reference power of 9 μCal/sec. The data were analyzed using Origin 7.0, MicroCal LLC ITC and fitted to a “one set of sites” model to obtain the stoichiometry (n), the thermodynamic association constant (Ka) and enthalpy energy ( H°).
    2.3. Synthesis of PDP-PEG-aldehyde
    The activation of the PDP-PEG-COOH carboxylic group was carried out as follows: the intermediate was solubilized in dichloromethane at a final concentration of 100 mg/ml, and a 2-fold molar excess of N,N′-dicyclohexylcarbodiimide (DCC) and hydroxybenzotriazole (HOBt) was added. After 1 h, a 3-fold molar excess of 4-aminobutyraldheyd-die-thylacetal was added. If necessary, the pH was adjusted to 8 with triethylamine. The reaction was allowed to proceed under stirring at room temperature overnight. The N,N-dicyclohexylurea was removed by filtration and the solution was dropped into diethyl-ether to pre-cipitate the product. After 1 h at −20 °C the product was recovered by filtration and dried under vacuum. The degree of diethyl acetal deri-vatization was determined by 1H NMR.
    The acetal was hydrolyzed at high temperature in acidic conditions to obtain the aldehyde group: a 50 mg of PEG was dissolved in 25 mM phosphate pH 2.15 at the final concentration of 50 mg/ml and main-tained at 60 °C for 2 h. The solution was used directly in the next con-jugation step with the protein.
    2.4. N-terminal pegylation of SpA and SpG
    The bacterial proteins were PEGylated with mPEG-aldehyde and with the synthesized PDP-PEG-aldehyde. Specifically, a 5 kDa MW
    polymer was used for SpA and a 20 kDa PEG was employed for SpG. Proteins were site-specifically modified at the N-terminus by con-ducting the PEGylation reaction under slightly acidic conditions (pH 4.5–5.5) as Fosfomycin calcium  described below. The protein concentration was determined by BCA assay or UV–Vis absorption at 280 nm.
    The conjugated proteins were characterized by SDS-PAGE, MALDI-TOF-TOF, RP-HPLC, FUV-CD; their affinity for IgG and the stoichio-metry of the complex were assessed by ITC and DLS.
    2.5. Synthesis of PEG5kDa-Nter-SpA and Cy5-PEG5kDa-Nter-SpA
    To a 2 mg/ml solution of SpA in 0.1 M sodium acetate buffer pH 5, PEG5kDa-aldehyde (5-fold molar excess) was firstly added and then, after 1 h, NaCNBH3 (100-fold molar excess) was added. The reaction mixture was allowed to proceed under stirring at room temperature, and its progress was monitored by RP-HPLC. After 24 h for PEG5kDa-aldehyde and after 96 h for PDP-PEG5kDa-aldehyde, the purification was performed by RP-HPLC with a Jupiter C18 column (250 × 4.6 mm, 300 Å, 5 μm; Phenomenex, USA) eluted with H2O + 0.1% TFA (eluent A) and ACN + 0.1% TFA (eluent B) at 1.0 ml/min flow-rate (gradient B
    %: 0′ 5%, 5′ 30%, 30′ 50%, 33′ 90%, 35′ 5%). The effluent was mon-itored by measuring the absorbance at 226 nm for analytical and at 280 nm for purification runs. The peak corresponding to the conjugate was collected and, after the acetonitrile (ACN) was removed under vacuum, the solution was lyophilized. PEG5kDa-SpA was used for in vitro characterization and binding studies to mAbs, and PDP-PEG5kDa-SpA was conjugated to Cy5 to perform FACS (fluorescence-activated cell