Protein a resin

The DNA fragment
of scFv fused with mouse IgG2a Fc by a glycine-serine
linker^{46 (link)} was ligated into the linearized
pFuse expression vector. HEK 293F cells (Thermo Scientific, R79007)
were cultured in shaker flasks containing FreeStyle medium (Thermo
Scientific, 12338026) and shaken at 125 rpm at 37 °C, with 5%
CO₂. Then, 2.5 × 10⁶ cells/mL were transfected
with the scFv-mFc expression plasmid mixed with PEI at a ratio of
1:2.5 (W/W). The expression media were harvested when the cell viability
decreased to below 75%. The collected supernatant was loaded onto
a column with Protein A resin (GenScript, L00210) twice, which was
pre-equilibrated in DPBS. After washing with 10 column volumes of
DPBS, the protein sample bound to the resin was eluted with elution
buffer (0.2 M glycine, 0.1 M NaCl, pH 2.5). Immediately after elution,
Tris-HCl (100 mM final concentration) was added to adjust the pH to
7.5. The eluted protein was then concentrated using Amicon Ultra centrifugal
filters (Merck Millipore, UFC903096), and the buffer was exchanged
with DPBS (pH 7.5) and further purified by size-exclusion chromatography
using a Superdex 200 increase 10/300 GL column (GE Healthcare, 10263259).
Purified protein aliquots were flash frozen by liquid nitrogen and
stored at −80 °C until use.

The recombinant fibre 1 protein was expressed in Escherichia coli. A gene fragment encoding the full-length fibre 1 protein was cloned into the prokaryotic expression vector, pET48a. The recombinant plasmid was transformed into BL21 (DE3) competent cells (TransGene) for inducible expression. The proteins were purified by Ni Sepharose Excel resin (GE Healthcare), following the manufacturer’s recommended protocol. The fibre 2 protein, the CAR protein, and the ECD, D1, and D2 domains of CAR were transiently expressed by transfecting Expi293F cells (Thermo Fisher Scientific) using Polyfectine (Sigma-Aldrich) [27 (link)]. Cell culture supernatants were harvested at 5 days post-transfection and centrifuged at 10,000 × g for 30 min to remove cell debris. The supernatants were sterile-filtered and purified with a protein A resin (GenScript Company). The expression of recombinant proteins was detected by SDS-PAGE and western blot analysis, using appropriate antibodies.

Primary antibodies 5C4 (specific to site Ø on pre-F conformation), 8C2 (binds to both the pre-F and post-F protein conformation) and 9F7 (specific to HEV, used here as negative control) were purified from the seroperitoneum of mice using Protein A Resin (GenScript, Piscataway, NJ, USA). We expressed 1129, AM14, AM22, 101F, MPE8 and Motavizumab using FreeStyle^TM 293-F cells (Gibco) via eukaryotic expression plasmid kindly donated by Jason S McLellan (Department of Molecular Biosciences College of Natural Sciences, The University of Texas at Austin, Austin, TX, USA).

For antibody purification, filtered supernatants were loaded onto a column packed with protein A resin (GenScript). After washing the column with PBS, Abs were eluted with 20 mM glycine pH 2.8 and neutralized with 1 M Tris HCl pH 9.5. Abs were concentrated and buffer exchanged to PBS using Ultra Centrifugal filter units (Merck). Concentrations of purified Abs were determined by NanoDrop.

DNA fragments encoding VH or VL of D27LEY were synthesized (IDT) and cloned into a vector derived from the pCEP4 vector (Invitrogen) which contains the CH1-CH2-CH3 sequence of IgG or the CL sequence of the kappa light chain. The resulting vectors, pCEP4(heavy) and pCEP4(k-light), were introduced into CHO-S cells (Gibco) at a density of 6x10⁶ cells/ml using ExpiFectamine (Gibco) to express D27LEY. The cells were grown in the ExpiCHO expression medium (Gibco) for 10 days. The culture supernatant was collected by centrifugation at 12,000g for 1 h at 4 ℃, diluted by half with Protein A binding buffer (PBS, pH 8.0), loaded onto an open column containing Protein A resin (Genscript), and eluted with Protein A elution buffer (0.1 M glycine, pH 3.0). The eluent was then immediately neutralized with Protein A neutralizing buffer (1M Tris-HCl, pH 8.5), and further purified using a HiLoad 26/60 Superdex 200 column (Cytiva) equilibrated with buffer A composed of 150 mM NaCl and 20 mM Tris-HCl (pH 8.0).