The production of recombinant platelet-derived growth factor D using the second generation modified vaccinia Ankara...

The vaccinia virus expression system is known for the efficient production of recombinant proteins with "appropriate" posttranslational modification using desired mammalian cell lines. However, being a replication competent virus, vaccinia virus poses a health threat to immunocompromised individuals and requires biosafety level 2 (BSL2) laboratory precautions, thereby restricting its use by the scientific community. Development of the host range restricted modified vaccinia Ankara (MVA) system has allowed researchers to work with a safer virus even at BSL1. Here, we report on the use of an improved second generation MVA viral system incorporating two selective markers and fluorescent proteins for easier recombinant virus identification. Notably, we demonstrate that this novel system is capable of producing secreted recombinant proteins, a finding not previously reported. Through purification and characterization of wild type and mutant platelet-derived growth factor D (PDGF D) dimer species, we demonstrate this system is capable of producing the latent full-length PDGF D dimer, partially processed intermediate dimer (hemidimer), as well as fully processed growth factor domain dimer that show chemical integrity and biological activity. Importantly, this system is amenable to scaling up for the mass production of recombinant PDGF D (rPDGF D) dimer species.