Insect Cells–Baculovirus System for the Production of Difficult to Express Proteins

Osz-Papai, Judit; Radu, Laura; Abdulrahman, Wassim; Kolb-Cheynel, Isabelle; Troffer-Charlier, N.; Birck, Catherine; Poterszman, Arnaud

doi:10.1007/978-1-4939-2205-5_10

Cited by 13 publications

(9 citation statements)

References 41 publications

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“…AcMNPV bacmids were purified using a Nucleobond PC500 DNA purification kit (740574 Macherey–Nagel) as described earlier 29 . Eluted DNA was precipitated with 0.7 volume of isopropanol, re-suspended at a concentration of 0.1 µg/µL and digested with Bsu36I enzyme (NEB) (Suppl.…”

Section: Methodsmentioning

confidence: 99%

HR-Bac, a toolbox based on homologous recombination for expression, screening and production of multiprotein complexes using the baculovirus expression system

Kolesnikova

Zachayus

Pichard

et al. 2022

Sci Rep

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The Baculovirus/insect cell expression system is a powerful technology for reconstitution of eukaryotic macromolecular assemblies. Most multigene expression platforms rely on Tn7-mediated transposition for transferring the expression cassette into the baculoviral genome. This allows a rigorous characterization of recombinant bacmids but involves multiple steps, a limitation when many constructs are to be tested. For parallel expression screening and potential high throughput applications, we have established an open source multigene-expression toolbox exploiting homologous recombination, thus reducing the recombinant baculovirus generation to a single-step procedure and shortening the time from cloning to protein production to 2 weeks. The HR-bac toolbox is composed of a set of engineered bacmids expressing a fluorescent marker to monitor virus propagation and a library of transfer vectors. They contain single or dual expression cassettes bearing different affinity tags and their design facilitates the mix and match utilization of expression units from Multibac constructs. The overall cost of virus generation with HR-bac toolbox is relatively low as the preparation of linearized baculoviral DNA only requires standard reagents. Various multiprotein assemblies (nuclear hormone receptor heterodimers, the P-TEFb or the ternary CAK kinase complex associated with the XPD TFIIH subunit) are used as model systems to validate the toolbox presented.

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Section: Methodsmentioning

confidence: 99%

HR-Bac, a toolbox based on homologous recombination for expression, screening and production of multiprotein complexes using the baculovirus expression system

Kolesnikova

Zachayus

Pichard

et al. 2022

Sci Rep

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“…Subsequently, viruses were generated by homologous recombination. Procedures followed to get insect cells expression are well documented [19,20].…”

Section: Protein Construct Preparationmentioning

confidence: 99%

Crystallization and Preliminary X-Ray Diffraction Analysis of a Mammal Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase

et al. 2017

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“…Such biochemistry experiments were instrumental in unraveling many fundamental processes, like the nature of triplet codons (Matthaei et al, 1962). But in many instances, the loss of context that occurs upon lysis or upon isolation incurs artifacts or loss of activity/structure, due to, for example, incorrect preparation techniques (Darling and Reid, 1979; Wang et al, 2011), or expression conditions (Osz-Papai et al, 2015). Furthermore, these assays also require large amounts of protein, thereby losing track of information on cell-to-cell variation, for example.…”

Section: The Need For Chemical Biology: Beyond Genetics and Biochemistrymentioning

confidence: 99%

Chemical Biology Gateways to Mapping Location, Association, and Pathway Responsivity

2019

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Here we discuss, how by applying chemical concepts to biological problems, methods have been developed to map spatiotemporal regulation of proteins and small-molecule modulation of proteome signaling responses. We outline why chemical-biology platforms are ideal for such purposes. We further discuss strengths and weaknesses of chemical-biology protocols, contrasting them against classical genetic and biochemical approaches. We make these evaluations based on three parameters: occupancy; functional information; and spatial restriction. We demonstrate how the specific choice of chemical reagent and experimental set-up unite to resolve biological problems. Potential improvements/extensions as well as specific controls that in our opinion are often overlooked or employed incorrectly are also considered. Finally, we discuss some of the latest emerging methods to illuminate how chemical-biology innovations provide a gateway toward information hitherto inaccessible by conventional genetic/biochemical means. Finally, we also caution against solely relying on chemical-biology strategies and urge the field to undertake orthogonal validations to ensure robustness of results.

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Insect Cells–Baculovirus System for the Production of Difficult to Express Proteins

Cited by 13 publications

References 41 publications

HR-Bac, a toolbox based on homologous recombination for expression, screening and production of multiprotein complexes using the baculovirus expression system

HR-Bac, a toolbox based on homologous recombination for expression, screening and production of multiprotein complexes using the baculovirus expression system

Crystallization and Preliminary X-Ray Diffraction Analysis of a Mammal Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase

Chemical Biology Gateways to Mapping Location, Association, and Pathway Responsivity

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