Production and Purification of Therapeutic Proteins

Therapeutic proteins and vaccines were produced as described [20 (link)]. Total RNA was isolated from B16F10 cells with RNeasy Mini Kit (Qiagen) and reverse transcribed with SuperScript III RT and random primers (Invitrogen) to synthesize B16F10 first-strand cDNA. eMLV env and gag sequences were amplified from B16F10 first-strand cDNA. eMLV env monomeric gp70 with a C-terminal His tag was cloned into the gWIZ vector (Gelantis) by Gibson assembly and produced by transiently transfecting HEK293F cells with the plasmid and polyethylenimine. The RBD was cloned by site-directed mutagenesis (NEB) and produced as gp70. Gag was expressed as a SUMO fusion using the pE-SUMOpro vector (LifeSensors) in Rosetta-gami 2 (DE) competent cells. Heavy and light chains of anti-env Abs were separately cloned into the gWIZ vector and anti-env Abs were produced by transiently co-transfecting HEK293F cells as above. His-tagged proteins were purified using TALON Metal Affinity Resin (Takara) and Abs were purified using rProtein A Sepharose Fast Flow resin (GE Healthcare). Endotoxin levels were below 0.1 total EU/dose as measured by the QCL-1000 chromogenic LAL assay (Lonza).