Rapid virus-free production of recombinant yellow fever virus envelope protein and its in-depth biophysical analysis.

The aim of this study was to develop a rapid and easy-to-adapt protocol for recombinant production of Orthoflavivirus proteins. We introduced the new-generation pOpiE2 vector for virus-free transient transfection of Trichopluisa ni High Five insect cells to produce the envelope protein ectodomain of Yellow Fever Virus Asibi and 17D strains. We achieved high yields in the range of 6–7 mg/L within five working days. In-depth biophysical analysis demonstrated highest protein purity, homogeneity, stability, and affinities to specific antibodies in the low nanomolar range. A combination of peptide mass fingerprint and intact mass spectrometry characterized the copurified precursor chaperone peptide pr and discovered a new posttranslational processing site for the recombinantly produced protein.