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

https://www.ncbi.nlm.nih.gov/pubmed/35089564

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.