Insect Cells-Baculovirus System for the Production of Difficult to Express Proteins: From Expression Screening for Soluble Constructs to Protein Quality Control.
Fiche publication
Date publication
janvier 2022
Journal
Methods in molecular biology (Clifton, N.J.)
Auteurs
Membres identifiés du Cancéropôle Est :
Dr BIRCK Catherine, Dr POTERSZMAN Arnaud, Mme KOLB-CHEYNEL Isabelle
Tous les auteurs :
Pichard S, Troffer-Charlier N, Kolb-Cheynel I, Poussin-Courmontagne P, Abdulrahman W, Birck C, Cura V, Poterszman A
Lien Pubmed
Résumé
Rapid preparation of proteins for functional and structural analysis is a major challenge both in academia and industry. The number potential targets continuously increases and many are difficult to express proteins which, when produced in bacteria, result in insoluble and/or misfolded recombinant proteins, protein aggregates, or unusable low protein yield. We focus here on the baculovirus expression vector system which is now commonly used for heterologous production of human targets. This chapter describes simple and cost-effective protocols that enable iterative cycles of construct design, expression screening and optimization of protein production. We detail time- and cost-effective methods for generation of baculoviruses by homologous recombination and titer evaluation. Handling of insect cell cultures and preparation of bacmid for cotransfection are also presented.
Mots clés
Baculovirus, Homologous recombination, Insect cells, Protein expression and production, Quality control
Référence
Methods Mol Biol. 2022 ;2406:281-317