![]() ![]() First, two strong promotors polyhedrin polH and p10 of the baculovirus Autographa californica multicapsid multinucleohedrovirus (AcMNPV) drive target protein expression to high levels, while the respective baculogenes are dispensible for virus propagation. The power of the baculoexpression vector system for heterologous protein expression has two reasons. ![]() Since its first publication more than 30 years ago, recombinant baculoviruses today are used for a broad range of applications, predominantly for heterologous protein expression in basic and applied research, for virus-like particles commercialized as vaccines and as gene delivery vectors for mammalian cells. In contrast to transient gene expression protocols, the required amount of DNA is minimal: 100 µg bacmid DNA is sufficient for a production scale of 10 L. This reduces the time from bacmid DNA to protein to eight days and reduces the risk of virus decay. In this study we show that Baculovirus generated by transfection-only is equally efficient in driving protein expression. The standard protocol for generating recombinant baculovirus comprises transfection of the bacmid followed by one or two subsequent virus amplification steps. We show that protein expression levels, purity and yield of the purified proteins are equally high for P0 and P1. To test if this P0 virus generated by bacmid transfection can be used directly for protein expression in either the screening or production process, we compared P0 versus amplified P1 virus-mediated protein expression. We demonstrate that transfection in suspension is as efficient as the standard protocol, but in addition allows generation of high amounts of P0 virus early in the process. We have then expressed and purified six different target proteins, among them four intracellular and two secreted proteins, by infecting insect cells either with P0 or P1 virus. In a first step we have adapted a recently published protocol that replaces the standard liposome-based transfection procedure of adherent insect cells by transfecting insect cells in suspension with a preformed DNA-PEI complex generating P0 virus. In this study we show that the conventional method to generate recombinant Baculovirus using a Tn7 transposition based system can be shortened to a single-step transfection-only procedure without further amplification. Despite the significant improvements introduced during the past years, the BEV system still has major drawbacks, primarily the time required to generate recombinant virus and virus instability for certain target proteins. In the last three decades, the Baculovirus expression vector system (BEV) has evolved to one of the most widely used eukaryotic systems for heterologous protein expression including approved vaccines and therapies. ![]()
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