A Time and Cost-Effective Pipeline for Expression Screening and Protein Production in Insect Cells Based on the HR-Bac Toolbox to Generate Recombinant Baculoviruses.
Fiche publication
Date publication
janvier 2024
Journal
Methods in molecular biology (Clifton, N.J.)
Auteurs
Membres identifiés du Cancéropôle Est :
Dr POTERSZMAN Arnaud
Tous les auteurs :
Loup-Forest J, Matuchet M, Schnitzler C, Pichard S, Poterszman A
Lien Pubmed
Résumé
The baculovirus expression vector system (BEVS) is recognized as a powerful platform for producing challenging proteins and multiprotein complexes both in academia and industry. Since a baculovirus was first used to produce heterologous human IFN-β protein in insect cells, the BEVS has continuously been developed and its applications expanded. We have recently established a multigene expression toolbox (HR-bac) composed of a set of engineered bacmids expressing a fluorescent marker to monitor virus propagation and a library of transfer vectors. Unlike platforms that rely on Tn7-medidated transposition for the construction of baculoviruses, HR-bac relies on homologous recombination, which allows to evaluate expression constructs in 2 weeks and is thus perfectly adapted to parallel expression screening. In this chapter, we detail our standard operating procedures for the preparation of the reagents, the construction and evaluation of baculoviruses, and the optimization of protein production for both intracellularly expressed and secreted proteins.
Mots clés
Baculovirus, Homologous recombination, Insect cells, Protein expression and production
Référence
Methods Mol Biol. 2024 ;2829:21-48