title: Multi-host expression system for recombinant production of challenging proteins.
abstract: Recombinant production of complex eukaryotic proteins for structural analyses typically requires a profound screening process to identify suitable constructs for the expression of ample amounts of properly folded protein. Furthermore, the evaluation of an optimal expression host has a major impact on protein yield and quality as well as on actual cost of the production process. Here we present a novel fast expression system for multiple hosts based on a single donor vector termed pFlp-Bac-to-Mam. The range of applications of pFlp-Bac-to-Mam comprises highly efficient transient transfection of HEK293-6E in serum-free suspension culture and subsequent large-scale production of challenging proteins expressing in mg per Liter level using either the baculoviral expression vector system or stable CHO production cell lines generated by Flp-mediated cassette exchange. The success of the multi-host expression vector to identify the optimal expression strategy for efficient production of high quality protein is demonstrated in a comparative expression study of three model proteins representing different protein classes: intracellular expression using a fluorescent protein, secretion of a single-chain-Fv-hIgG1Fc fusion construct and production of a large amount of highly homogeneous protein sample of the extracellular domain of a Toll-like receptor. The evaluation of the production efficiency shows that the pFlp-Bac-to-Mam system allows a fast and individual optimization of the expression strategy for each protein class.
authors.affiliation: Department of Molecular Structural Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Steffen
authors.initials: S
authors.lastname: Meyer
authors.forename: Carmen
authors.initials: C
authors.lastname: Lorenz
authors.forename: Bahar
authors.initials: B
authors.lastname: Baser
authors.forename: Mona
authors.initials: M
authors.lastname: Wördehoff
authors.forename: Volker
authors.initials: V
authors.lastname: Jäger
authors.forename: Joop
authors.initials: J
authors.lastname: van den Heuvel
mesh_terms: D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D003412:Cricetulus; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism*
chemical_list: D004274:DNA, Recombinant; D011994:Recombinant Proteins