ArticleBMC biotechnology2017
A new single-step protocol for rapid baculovirus-driven protein production in insect cells.
Article in BMC biotechnology, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 33 papers.
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33 citing papers in PubMed, 52 citations in OpenAlex.
- Steady expression of heterologous genes in insect cells based on rebuilt Bacmid vectors.Biotechnology letters · 2026Article
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- Harnessing Transient Expression Systems with Plant Viral Vectors for the Production of Biopharmaceuticals inInternational journal of molecular sciences · 2025Review
- Rapid and Economic Baculovirus Titer Determination Using a Novel Transgenic Sf9-QE Cell Line.Insects · 2025Article
- Real time characterization of the MAPK pathway using native mass spectrometry.Communications biology · 2025Article
- Equi-MOI ratio for rapid baculovirus-mediated multiprotein co-expression in insect cells integrating selenomethionine for structural studies.FEBS open bio · 2025Article
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- Cardenolides in the defensive fluid of adult large milkweed bugs have differential potency on vertebrate and invertebrate predator NaRoyal Society open science · 2024Article
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- Application of Protein Expression inScientifica · 2024Review
- Native mass spectrometry of proteoliposomes containing integral and peripheral membrane proteins.Chemical science · 2023Article
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- Activity, substrate preference and structure of the HsMCM8/9 helicase.Nucleic acids research · 2023Article
- Screening of Membrane Protein Production by Comparison of Transient Expression in Insect and Mammalian Cells.Biomolecules · 2023Article
- BICD2 phosphorylation regulates dynein function and centrosome separation in G2 and M.Nature communications · 2023Article
- Recombinant Protein Production Using the Baculovirus Expression Vector System (BEVS).Methods in molecular biology (Clifton, N.J.) · 2023Article
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundIn 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. 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 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.
methodsIn 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. 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.
resultsWe 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. 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 show that protein expression levels, purity and yield of the purified proteins are equally high for P0 and P1.
conclusionThe standard protocol for generating recombinant baculovirus comprises transfection of the bacmid followed by one or two subsequent virus amplification steps. In this study we show that Baculovirus generated by transfection-only is equally efficient in driving protein expression. This reduces the time from bacmid DNA to protein to eight days and reduces the risk of virus decay. 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.
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