Significant Productivity Improvement of the Baculovirus Expression Vector System by Engineering a Novel Expression Cassette

Yang

Pest Management Science

et al. 2019

BACKGROUND Baculoviruses provide long‐lasting control of crop pests and are harmless to humans and non‐target animals, making them attractive bioinsecticides. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has a wide‐host range and is one such commercial bioinsecticide, but its low infectivity to older larvae and less‐sensitive species precludes its large‐scale application. We sought to improve the infectivity of AcMNPV. RESULTS Two enhancing factors, the truncated enhancin from Agrotis segetum granulovirus and GP37 from Cydia pomonella granulovirus, were expressed in fusion with the N‐terminal and middle domain of the polyhedrin envelope protein of AcMNPV. Western blotting and immunoelectron microscopy analysis indicated that the enhancing factors were expressed on the occlusion bodies of the resulting AcMNPV variants. Bioassays showed that the median lethal doses of the recombinant viruses were 3.9‐fold to 7.4‐fold lower than those of the wild‐type virus against the second and fourth instar of Spodoptera exigua larvae. The yields of occlusion bodies from the two recombinants in S. exigua larvae were comparable with those of the wild‐type virus both in vitro and in vivo. Further bioassays showed that the AcMNPV variants fusing the enhancing factors were incapable of infecting the second instar larvae of S. litura, Helicoverpa armigera, and Pyrausta nubilalis, which were not sensitive to the wild‐type AcMNPV. CONCLUSION These genetically modified AcMNPV variants exhibited an enhanced infectivity and may offer better baculovirus control of crop pests. © 2019 Society of Chemical Industry

Section: Discussionsupporting

confidence: 85%

Displaying enhancing factors on the surface of occlusion bodies improves the insecticidal efficacy of a baculovirus

Yang

Pest Management Science

et al. 2019

“…17,18 G omez-Sebasti an et al engineered a novel expression cassette containing various baculovirus genomic elements such as transactivators IE1 and IE0 and enhancer sequences. 18 Insect cells infected with those viruses showed increased cell viability and integrity after infection, and an increase in recombinant protein yields. A similar effect was achieved when the baculovirus apoptotic inhibitor P35 was constitutively expressed from the insect cells.…”

Section: Introductionmentioning

confidence: 99%

Improving the baculovirus expression vector system with vankyrin‐enhanced technology

Steele

Stone

Franklin

et al. 2017

Biotechnology Progress

The baculovirus expression vector system (BEVS) is a widely used platform for the production of recombinant eukaryotic proteins. However, the BEVS has limitations in comparison to other higher eukaryotic expression systems. First, the insect cell lines used in the BEVS cannot produce glycoproteins with complex-type N-glycosylation patterns. Second, protein production is limited as cells die and lyse in response to baculovirus infection. To delay cell death and lysis, we transformed several insect cell lines with an expression plasmid harboring a vankyrin gene (P-vank-1), which encodes an anti-apoptotic protein. Specifically, we transformed Sf9 cells, Trichoplusia ni High Five™ cells, and SfSWT-4 cells, which can produce glycoproteins with complex-type N-glycosylation patterns. The latter was included with the aim to increase production of glycoproteins with complex N-glycans, thereby overcoming the two aforementioned limitations of the BEVS. To further increase vankyrin expression levels and further delay cell death, we also modified baculovirus vectors with the P-vank-1 gene. We found that cell lysis was delayed and recombinant glycoprotein yield increased when SfSWT-4 cells were infected with a vankyrin-encoding baculovirus. A synergistic effect in elevated levels of recombinant protein production was observed when vankyrin-expressing cells were combined with a vankyrin-encoding baculovirus. These effects were observed with various model proteins including medically relevant therapeutic proteins. In summary, we found that cell lysis could be delayed and recombinant protein yields could be increased by using cell lines constitutively expressing vankyrin or vankyrin-encoding baculovirus vectors.

“…The problem with these systems, however, is that it is difficult to produce some glycoproteins in sufficient quantities. In most of the previous studies, therefore, focused on the improvement of host glycosylation pathway and baculovirus vector to increase protein production in both quantity and quality [2][3][4].…”

Section: Introductionmentioning

confidence: 99%

Roles of silkworm endoplasmic reticulum chaperones in the secretion of recombinant proteins expressed by baculovirus system

Imai

Kusakabe

et al. 2015

Mol Cell Biochem

Baculovirus expression vector system (BEVS) is widely used for production of recombinant eukaryotic proteins in insect larvae or cultured cells. BEVS has advantages over bacterial expression system in producing post-translationally modified secreted proteins. However, for some unknown reason, it is very difficult for insects to secrete sufficiently for certain proteins of interest. To understand the reasons why insect cells fail to secrete some kinds of recombinant proteins, we here employed three mammalian proteins as targets, EPO, HGF, and Wnt3A, with different secretion levels in BEVS and investigated their mRNA transcriptions from the viral genome, subcellular localizations, and interactions with silkworm ER chaperones. Moreover, we observed that no significantly influence on the secretion amounts of all three proteins when depleting or overexpressing most endogenous ER chaperone genes in cultured silkworm cells. However, among all detected ER chaperones, the depletion of BiP severely decreased the recombinant protein secretion in BEVS, indicating the possible central role of Bip in silkworm secretion pathway.