title: An optimized expression vector for improving the yield of dengue virus-like particles from transfected insect cells.
abstract: Recombinant virus-like particles (rVLPs) of flaviviruses are non-infectious particles released from cells expressing the envelope glycoproteins prM and E. Dengue virus rVLPs are recognized as a potential vaccine candidate, but large scale production of these particles is hindered by low yields and the occurrence of cytopathic effects. In an approach to improve the yield of rVLPs from transfected insect cells, several components of a dengue serotype 2 virus prM+E expression cassette were modified and the effect of these modifications was assessed during transient expression. Enhancement of extracellular rVLP levels by simultaneous substitutions of the prM signal peptide and the stem-anchor region of E with homologous cellular and viral counterparts, respectively, was further augmented by codon optimization. Extensive formation of multinucleated cells following transfection with the codon-optimized expression cassette was abrogated by introducing an E fusion loop mutation. This mutation also helped restore the extracellular E levels affected negatively by alteration of a charged residue at the pr-M junction, which was intended to promote maturation of rVLPs during export. Optimized expression cassettes generated in this multiple add-on modification approach should be useful in the generation of stably expressing clones and production of dengue virus rVLPs for immunogenicity studies.
authors.affiliation: Center for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand.
authors.forename: Nicha
authors.initials: N
authors.lastname: Charoensri
authors.affiliation: Medical Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok 10700, Thailand.
authors.forename: Amporn
authors.initials: A
authors.lastname: Suphatrakul
authors.affiliation: Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand.
authors.forename: Rungtawan
authors.initials: R
authors.lastname: Sriburi
authors.affiliation: Medical Science Research Equipment Center, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand.
authors.forename: Thippawan
authors.initials: T
authors.lastname: Yasanga
authors.affiliation: Department of Microbiology, Faculty of Public Health, Mahidol University, Bangkok 10400, Thailand.
authors.forename: Jiraphan
authors.initials: J
authors.lastname: Junjhon
authors.affiliation: Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand.
authors.forename: Poonsook
authors.initials: P
authors.lastname: Keelapang
authors.affiliation: Research Institute for Health Sciences, Chiang Mai University, Chiang Mai 50200, Thailand.
authors.forename: Utaiwan
authors.initials: U
authors.lastname: Utaipat
authors.affiliation: Medical Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok 10700, Thailand; Dengue Hemorrhagic Fever Research Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
authors.forename: Chunya
authors.initials: C
authors.lastname: Puttikhunt
authors.affiliation: Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand; Biomedical Technology Research Center, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency at the Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand.
authors.forename: Watchara
authors.initials: W
authors.lastname: Kasinrerk
authors.affiliation: Medical Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok 10700, Thailand; Dengue Hemorrhagic Fever Research Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
authors.forename: Prida
authors.initials: P
authors.lastname: Malasit
authors.affiliation: Medical Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok 10700, Thailand; Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand. Electronic address: nsittiso@mail.med.cmu.ac.th.
authors.forename: Nopporn
authors.initials: N
authors.lastname: Sittisombut
mesh_terms: D000818:Animals; D002460:Cell Line; D003062:Codon / Q000235:genetics; D003715:Dengue / Q000517:prevention & control* / Q000821:virology; D053059:Dengue Vaccines; D003716:Dengue Virus / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D015870:Gene Expression; D005822:Genetic Vectors; D006023:Glycoproteins; D006801:Humans; D007313:Insecta; D021382:Protein Sorting Signals / Q000235:genetics; D014162:Transfection; D058425:Vaccines, Virus-Like Particle; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D003062:Codon; D053059:Dengue Vaccines; D006023:Glycoproteins; D021382:Protein Sorting Signals; D058425:Vaccines, Virus-Like Particle; D014759:Viral Envelope Proteins; C074932:prM protein, Flavivirus
keywords: Dengue virus; Insect cell lines; Transfection; Vaccine candidate; Virus-like particle