Recombinant EBV Glycoprotein Production

The DNA sequence encoding EBV gL (residues 24 to 137; UniProt ID G3CKR4) linked to EBV gH (residues 19 to 678; UniProt ID G3CKS5; C-terminal 6× His tag) with a (G4S)3 linker and the DNA sequence encoding EBV gB (residues 25 to ∼683, UniProt ID R4R670) were cloned into pcDNA3.1. A CD5 signal peptide DNA sequence was inserted into the N terminus of genes of interest. Antibodies against EBV gHgL, E1D1 (45 (link)), and AMMO1 (60 (link)) and recombinant gHgL and gB were expressed in 293F cells. The supernatant was collected and purified by Ni⁺ affinity chromatography (Ni Sepharose Excel; Cytiva). For gHgL, the eluate was concentrated and purified by size exclusion chromatography (SEC) (Superdex 200 Increase 10/300 GL; Cytiva) running at 50 mM NaCl and 50 mM HEPES, pH 7.5; fractions were collected and applied to anion-exchange chromatography (HiTrap Capto Q; 5 mL; Cytiva) and eluted by linear ion-strength increment; fractions were concentrated and purified by SEC (Superdex 200 Increase 10/300 GL) running at 50 mM HEPES and 150 mM NaCl, pH 7.5. For gB, the eluate was concentrated and purified by SEC (Superdex 200 Increase 10/300 GL; Cytiva) running at 50 mM NaCl and 150 mM HEPES, pH 7.5. For E1D1 and AMMO1, the supernatant was captured by protein A beads (L00210; GenScript) followed by SEC (Superdex 200 Increase 10/300 GL; Cytiva) running in PBS.