FlexiBAC: a versatile, open-source baculovirus vector system for protein expression, secretion, and proteolytic processing

Régis P Lemaitre,David N Drechsel,Barbara Borgonovo,Aliona Bogdanova,Jeffrey B Woodruff

doi:10.1186/s12896-019-0512-z

Abstract

BackgroundBaculovirus-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.ResultsTo overcome the limitations of traditional baculovirus expression systems, we engineered “FlexiBAC”. This system allows recombinant baculovirus formation inside insect cells and reduces the time between initial cloning and protein production to 13 days. FlexiBAC includes 143 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. This system also overexpresses recombinant furin convertase to allow efficient proteolytic processing of secreted proteins. We demonstrate that FlexiBAC 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.ConclusionsFlexiBAC is a protein expression system for production of both cytosolic proteins and secreted proteins that require proteolytic maturation. The design of FlexiBAC and its expansive complementary shuttle vector system reduces cloning steps and simplifies baculovirus production.

Highlights

Baculovirus-mediated expression in insect cells is a powerful approach for protein production
We demonstrate that FlexiBAC is suited for the expression of proteins deemed difficult to reconstitute, including mature Transforming growth factor Beta (TGF-β) family growth factors, intrinsically disordered proteins, as well as proteins containing numerous coiled-coil domains
Overview of the FlexiBAC system To streamline the production of viral stocks, we engineered a bacmid encoding a replication-defective baculovirus, which we term “defective baculovirus genome (DefBac)” (Fig. 1)

Summary

Introduction

Baculovirus-mediated expression in insect cells is a powerful approach for protein production. Insect cells can grow in serum-free media, which greatly facilitates purification of secreted proteins from the conditioned media Despite these advantages, recombinant protein production using baculovirus is time-consuming. To make baculovirus production simpler and faster, more recent protocols have facilitated production of recombinant baculovirus genomes using site-specific transposition in E. coli (Bac-to-Bac®, Thermo Fisher Scientific) or homologous recombination in insect cells (flashBACTM, Oxford Expression Technologies). Both systems employ the Lemaitre et al BMC Biotechnology (2019) 19:20 polyhedrin promoter to drive high-level, late expression of the target protein(s) of interest. As reconstituted systems are analyzed more and more via light microscopy, there is a pressing need for more fluorophore tagging options

Methods

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Discussion

Conclusion