HR-Bac, a toolbox based on homologous recombination for expression, screening and production of multiprotein complexes using the baculovirus expression system - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue Scientific Reports Année : 2022

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

Résumé

Abstract 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.
Fichier principal
Vignette du fichier
SciRep2022.pdf (3.69 Mo) Télécharger le fichier
PDF Datastream.pdf (661.92 Ko) Télécharger le fichier
Origine : Fichiers éditeurs autorisés sur une archive ouverte
Licence : CC BY - Paternité

Dates et versions

hal-03860882 , version 1 (18-11-2022)

Identifiants

Citer

Olga Kolesnikova, Amélie Zachayus, Simon Pichard, Judit Osz, Natacha Rochel, et al.. HR-Bac, a toolbox based on homologous recombination for expression, screening and production of multiprotein complexes using the baculovirus expression system. Scientific Reports, 2022, 12 (1), pp.2030. ⟨10.1038/s41598-021-04715-5⟩. ⟨hal-03860882⟩
183 Consultations
37 Téléchargements

Altmetric

Partager

Gmail Facebook X LinkedIn More