Enhancement of exogenous protein stability in AcMNPV by overexpressing lef5 gene during passaging

The baculovirus insect cell expression system is pivotal for exogenous protein expression. However, serial passages of baculovirus in insect cells often result in defective virus generation and a rapid decline in exogenous protein expression, limiting its wider application. Previous studies have shown that the expression of the late expression factor 5 (lef5) from other baculoviruses can enhance the stability of exogenous genes in Autographa californica multiple nucleopolyhedrovirus (AcMNPV). In this study, we engineered diverse recombinant strains of AcMNPV to express the enhanced green fluorescent protein (eGFP) and enterovirus 71 virus-like particles (EV71-VLPs), co-expressed with the P1 and 3CD proteins of EV71. We investigated the influence of lef5 overexpression, regulated by various promoters, on the stability of exogenous genes and their protein expressions. Notably, lef5 overexpression significantly improved the stability of eGFP and EV71-P1 gene and protein expressions during serial passages in Sf9 cells. Specifically, the lef5 overexpression driven by the op166 promoter was more beneficial for enhancing the expression stability of complex exogenous proteins, such as EV71-VLPs. This study underscores the importance of lef5 overexpression in maintaining the stability of exogenous gene expression in baculovirus systems, particularly for producing complex proteins such as VLPs of enterovirus. KEY POINTS: • The overexpression of lef5 stabilizes exogenous gene expression in the baculovirus system. • Promoter choice affects lef5-mediated gene expression stability. • The overexpression of lef5 is crucial for producing complex proteins like EV71-VLPs.