The baculovirus expression vector system (BEVS) is well known for its ability to produce large amounts of recombinant proteins with eukaryotic modifications. Scientists all over the world have used this system to produce thousands of different recombinant proteins, including many glycoproteins, facilitating research on their structure, function, and roles in human disease. Many biotechnology companies use the BEVS for basic research and/or to produce recombinant proteins for clinical use as vaccines, therapeutics, or diagnostic reagents, and BEVS-manufactured clinical products are clearly on the horizon. However, our relatively poor understanding of insect cell protein processing pathways and their apparent inability to routinely produce recombinant glycoproteins with authentic oligosaccharide side chains are major problems with this system. For the past 19 years, we have studied the molecular mechanisms of protein processing in insect cells. For the past decade, most of our work has focused on protein glycosylation. Through a combination of basic and applied research, we have contributed a better understanding of insect protein /V-glycosylation pathways and produced new insect cell-baculovirus expression systems with improved glycoprotein processing capabilities. In this competing renewal application, we propose to build upon these accomplishments. One general goal will be to extend our previous efforts to metabolically engineer the protein /V-glycosylation pathway of lepidopteran insect cells by transformation with mammalian genes. This is important because additional engineering is needed to produce transgenic insect cell lines with more extensively humanized A/-glycosylation pathways. These cell lines will be broadly useful to biomedical researchers as improved hosts for baculovirus-mediated recombinant /V-glycoprotein production. The other general goal will be to extend our previous efforts to understand the basic nature of the endogenous protein A/-glycosylation pathway in lepidopteran insect cells. We will use metabolic engineering as a powerful and innovative approach to obtain new information on this pathway. We also will continue to clone the genes encoding endogenous protein /V-glycosylation functions and characterize their products. The results will provide a more detailed understanding of insect protein /V-glycosylation mechanisms at the molecular level. This is important because protein /V-glycosylation is a major biosynthetic pathway in all eukaryotes, but it is relatively poorly understood in insect systems. In addition to providing fundamental support for the BEVS, a better understanding of the similarities and differences between insect and mammalian protein /V-glycosylation pathways could reveal ways to interrupt disease transmission by insect vectors or to control agriculturally important insect pests, which would have a major impact on human health.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Vector Biology Study Section (VB)
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Marino, Pamela
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University of Wyoming
Schools of Earth Sciences/Natur
United States
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Harrison, Robert L; Jarvis, Donald L (2016) Transforming Lepidopteran Insect Cells for Continuous Recombinant Protein Expression. Methods Mol Biol 1350:329-48
Serena, María Soledad; Geisler, Christoph; Metz, Germán Ernesto et al. (2016) Production of pseudorabies virus recombinant glycoprotein B and its use in an agar gel immunodiffusion (AGID) test for detection of antibodies with sensitivity and specificity equal to the virus neutralization assay. J Virol Methods 230:9-12
Harrison, Robert L; Jarvis, Donald L (2016) Transforming Lepidopteran Insect Cells for Improved Protein Processing and Expression. Methods Mol Biol 1350:359-79
Mabashi-Asazuma, Hideaki; Sohn, Bong-Hee; Kim, Young-Soo et al. (2015) Targeted glycoengineering extends the protein N-glycosylation pathway in the silkworm silk gland. Insect Biochem Mol Biol 65:20-7
Geisler, Christoph; Mabashi-Asazuma, Hideaki; Kuo, Chu-Wei et al. (2015) Engineering β1,4-galactosyltransferase I to reduce secretion and enhance N-glycan elongation in insect cells. J Biotechnol 193:52-65
Mabashi-Asazuma, Hideaki; Kuo, Chu-Wei; Khoo, Kay-Hooi et al. (2014) A novel baculovirus vector for the production of nonfucosylated recombinant glycoproteins in insect cells. Glycobiology 24:325-40
Lin, Chi-Hung; Kuo, Chu-Wei; Jarvis, Donald L et al. (2014) Facile removal of high mannose structures prior to extracting complex type N-glycans from de-N-glycosylated peptides retained by C18 solid phase to allow more efficient glycomic mapping. Proteomics 14:87-92
Toth, Ann M; Kuo, Chu-Wei; Khoo, Kay-Hooi et al. (2014) A new insect cell glycoengineering approach provides baculovirus-inducible glycogene expression and increases human-type glycosylation efficiency. J Biotechnol 182-183:19-29
Serena, María Soledad; Geisler, Christoph; Metz, Germán Ernesto et al. (2013) Expression and purification of Suid Herpesvirus-1 glycoprotein E in the baculovirus system and its use to diagnose Aujeszky's disease in infected pigs. Protein Expr Purif 90:1-8
Lin, Chi-Hung; Jarvis, Donald L (2013) Utility of temporally distinct baculovirus promoters for constitutive and baculovirus-inducible transgene expression in transformed insect cells. J Biotechnol 165:11-7

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