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PLoS One. 2013 Jul 17;8(7):e68674. doi: 10.1371/journal.pone.0068674. Print 2013.

Multi-host expression system for recombinant production of challenging proteins.

Author information

1
Department of Molecular Structural Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.

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.

PMID:
23874717
PMCID:
PMC3714276
DOI:
10.1371/journal.pone.0068674
[Indexed for MEDLINE]
Free PMC Article

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