RESUMEN
Stable mammalian production cell lines in suspension culture enable the reproducible expression of target genes in any desired scale using bioreactor technology. Targeted integration methods have been developed to cut down timelines for the generation of stable producer cell lines. The powerful Flp recombinase mediated cassette exchange (RMCE) technique allows fast integration of target genes in preselected and optimized high expression loci in so called master cell lines. Up to now, these cells only enable the expression from a single locus. Here, we describe the set-up required for the generation of multiple tagged master cell lines on the example of a binary RMCE expression system in the glycosylation mutant CHO Lec3.2.8.1 cell line. We show how this technology is used for the expression of proteins from multiple loci by generating a binary RMCE expression system. The tools and strategy for the construction of binary master cell lines with different combinations of expression level are described in detail. The binary production cell lines show independent expression of the individual exchange loci of the producer cell lines. The expression level for the model protein tdTomato is the cumulative expression for the chosen combination of the expression loci of the master cell line. This binary RMCE expression system can be further developed to a multi RMCE expression system for co-expression of protein complex subunits with predetermined expression ratio of each individual exchange locus.
Asunto(s)
Clonación Molecular/métodos , Sitios Genéticos , Vectores Genéticos/química , Genoma , Transgenes , Animales , Células CHO , Línea Celular , Cricetulus , ADN Nucleotidiltransferasas/genética , ADN Nucleotidiltransferasas/metabolismo , Efecto Fundador , Expresión Génica , Vectores Genéticos/metabolismo , Genómica/métodos , Plásmidos/química , Plásmidos/metabolismo , Recombinación Genética , Transformación GenéticaRESUMEN
In this chapter conventional and emerging new technologies for the production of complex biologics in mammalian expression systems are summarized. The essential features of the most relevant methods to generate stable production cell lines for the expression of recombinant multi-protein complexes are described. Especially the promising multiple targeted integration strategy by Flp or CRISPR/Cas9 mediated recombination and their future impact on multi-protein expression are highlighted.
Asunto(s)
Ingeniería de Proteínas/métodos , Proteínas Recombinantes/biosíntesis , Animales , Sistemas CRISPR-Cas , Técnicas de Cultivo de Célula , Línea Celular , ADN Nucleotidiltransferasas/genética , ADN Nucleotidiltransferasas/metabolismo , Regulación de la Expresión Génica , Vectores Genéticos , Humanos , Complejos Multiproteicos , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Subunidades de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Relación Estructura-Actividad , Transcripción Genética , TransfecciónRESUMEN
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.