Kta pure fast protein liquid chromatography system

LAG‐3:Fc sample was gel filtrated into fresh TBSB buffer the day of SPR experiments using a Superdex S200 10/300 column and ÄKTA Pure fast protein liquid chromatography system (GEHealthcare Life Sciences). Analysis of LAG‐3:Fc‐pHLA‐DR1 binding was performed on a BIAcore T200 instrument (GE Healthcare Life Sciences). All experiments were performed at 25°C in TBSB buffer. Prepared biotinylated pHLA‐DR molecules were immobilized to covalently linked streptavidin coated CM5 sensor chips prepared as previously described [42]. Biotinylated pHLA‐DR molecules were bound to the chip surface at a flow rate of 10 μL/min. LAG‐3:Fc molecules were twofold serially diluted and sequentially injected at 30 μL/min (30 s association, 300 s dissociation) over the pHLA‐DR chip surface. Recorded sensograms were reference subtracted against a control flow cell of HLAA*02:01‐hTERT_540‐548 after confirmation as a suitable non‐binding control ligand. Sensograms were analyzed using BIAevaluation version 4.1 (GE Healthcare Life Sciences) and plotted using GraphPad Prism version 5 (GraphPad Software, Inc). Kinetic analyses of LAG‐3:Fc binding were performed using the simultaneous k_on/k_off fitting function of the binding model specified. Equilibrium analyses were performed using nonlinear regression least squares ordinary fit of the one‐site specific binding model using GraphPad Prism version 5.

A human codon optimized HCV immunogen was inserted into the donor vector between the cytomegalovirus promoter and bovine growth hormone poly‐A sequence by In‐Fusion cloning to generate the shuttle vector. The shuttle vector was cloned into the ChAdOx1 destination vector, using ThermoFisher’s LR gateway cloning method, and linearized using PmeI restriction and transfected into T‐REx‐293 cells (ThermoFisherScientific) to generate ChAdOx1 vaccines. For MVA vaccines, the HCV immunogen was cloned at the F11 locus of the pMVA‐shuttle vector and the recombinant MVA generated by recombining the pMVA‐shuttle vector and WT MVA. HCV viral vector vaccines were manufactured by the Viral Vector Core Facility (Jenner Institute, University of Oxford, Oxford, UK).
Recombinant proteins were expressed using the Freestyle 293‐F cells stable transfected cell clone, purified from the culture supernatants using cobalt‐charged TALON metal affinity resin (Clontech) by the C‐terminal polyhistidine tag followed by size‐exclusion chromatography on a Superdex200 prep grade 16/600 column (GE Healthcare), using the ÄKTA pure fast protein liquid chromatography system (GE Healthcare) equilibrated in PBS (pH 6.8). Fractions containing the HMW species were pooled and concentrated through an Amicon® 30‐kDa molecular‐weight cut‐off ultracentrifugal device (Merck).

The FLAG-tagged hTAS1R2 samples that had been eluted from the ANTI-FLAG M2 beads were pooled and concentrated to 0.3–0.5 mg/mL using a 30-kDa MWCO filter column (Vivaspin, Sartorius, Aubagne, France). Then the concentrated FLAG-tagged hTAS1R2 was purified by gel filtration as previously reported^{36 (link)}. The samples were then loaded for gel filtration chromatography (Superdex 200 Increase 10/300GL column) on an Äkta Pure fast protein liquid chromatography system (GE Healthcare, Velizy-Villacoublay, France). The column was equilibrated with 2 column volumes of wash buffer (PBS, 0.1% LMNG, pH 7.3) before the immunopurified FLAG-tagged hTAS1R2 sample was applied. After loading, the column was rinsed with wash buffer at 0.5 mL/min, and the column flow through was monitored by UV absorbance at 280 nm. The molecular masses of the FLAG-tagged hTAS1R2-detergent complexes were estimated by calibrating the column with a gel filtration standard mixture (Sigma-Aldrich). The following standard proteins were used: thyroglobulin (669 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), monomeric BSA (66 kDa), carbonic anhydrase (29 kDa), myoglobin (17 kDa) and lysozyme (14.3 kDa). The protein fractions (0.5 mL) were collected using an automated fraction collector. The collected fractions were deposited on SDS-PAGE, stained by Coomassie blue and subjected to immunoblotting analysis.