A Baculovirus Expression Vector System for Simultaneous Protein Expression in Insect and Mammalian Cells

Abstract

Since the number of potential drug targets identified has significantly increased in the past decade, rapid expression of recombinant proteins in sufficient amounts for structure determination and modern drug discovery is one of the major challenges in pharmaceutical research. As a result of its capacity for insertion of large DNA fragments, its high yield of recombinant protein and its high probability of success compared to protein expression in Escherichia coli, the baculovirus expression vector system (BEVS) is used routinely to produce recombinant proteins in the milligram scale. For some targets, however, expression of the recombinant protein with the BEVS in insect cells fails and mammalian expression systems have to be used to achieve proper post-translational processing of the nascent polypeptide. We now introduce a modified BEVS as a very useful tool for simultaneously testing the expression of target proteins in both insect and mammalian cells by using baculovirus infection of both host systems. The expression yields in insect cells are comparable to those obtained with state-of-the-art baculovirus vectors, such as the Bac-to-Bac system. Using the same virus, we can transduce mammalian cells to quickly assess target gene expression feasibility and optimize expression conditions, eliminating additional cloning steps into mammalian expression vectors. This reduces time and effort for finding appropriate expression conditions in various hosts.

Assembly defects of the human tRNA splicing endonuclease contribute to impaired pre-tRNA processing in pontocerebellar hypoplasia

“…To gain functional insights into human TSEN/CLP1, we designed an expression vector series based on the MultiBac system 32 that allows combinatorial protein complex production in insect and mammalian cells by utilizing a CMV/p10 dual promoter 33 (Fig. 1a,b and Extended Data Fig.…”

Recent advances in the production of proteins in insect and mammalian cells for structural biology

“…Traditional plaque assays require experience and are time-consuming and so do not fit into a streamlined workflow. This has led to the development of alternative methods, including end-point dilution using fluorescence from GFP co-expressed with the protein of interest (Philipps et al, 2005), measuring infection by monitoring cell viability using the sensitive redox indicator AlamarBlue™ and more indirect approaches based on quantitative polymerase chain reaction (Q-PCR) (Hitchman et al, 2007;Lo and Chao, 2004). Recently the accuracy of all of the available methods for estimating baculovirus titres has been compared and, with the exception of Q-PCR, shown to give similar values for viral titre (Roldao et al, 2009).…”

Expression of recombinant proteins in plants by using baculovirus vectors

“…The insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) is a rod-shaped, enveloped virus with a double-stranded genomic DNA (Beljelarskaya et al, 2002;Kost and Condreay, 2002;Van Loo et al, 2001). Compared to other viral vector systems, baculovirus vectors offer many advantages such as the ability of large DNA fragment insertion, proper protein folding, and post-translational modifications for comparable biological activity (Cameron and Bishop, 1989;Jarvis et al, 1996;King and Possee, 1992;Luckow and Summers, 1988;Miller et al 1988;Philipps et al, 2005). A large variety of foreign genes have been expressed in different baculovirus expression vectors, which primarily utilize an effective promoter of the very late gene polh or p10 (Patel et al, 1992;Possee, 1997).…”