FlexiBAC: a Versatile, Open-Source Baculovirus Vector System for Protein Expression, Secretion, and Proteolytic Processing

ABSTRACTBaculovirus-mediated expression in insect cells is a powerful approach for protein production. However, many existing methods are time consuming, offer limited options for protein tagging, and are unsuitable for secreted proteins requiring proteolytic maturation, such as TGF-β family growth factors. To overcome these limitations, we engineered “FlexiBAC”, a system that simplifies baculovirus production and permits furin-driven proteolytic maturation of targets. This system allows recombinant baculovirus formation inside insect cells and reduces the time between initial cloning and protein production to 13 days. FlexiBAC includes 146 shuttle vectors that append combinations of purification tags, fluorescent markers, proteolytic cleavage sites, trafficking signals, and chemical conjugation tags to the termini of the target protein. We demonstrate that this system can be used to produce high levels of mature, active forms of TGF-β family growth factors, such as Activin A, as well as other proteins that are typically difficult to reconstitute, such as proteins rich in coiled-coil, low complexity, and disordered domains.