MTLR2 in the BEVS. However, capturing ECD-mTLR2 from cell culture supernatants of the CHO get ML 281 producer cell lines provided a higher purity with less contamination compared to the POR 8 web expression in BEVS, where a significant contamination by host cell proteins is observed due to cell lysis (Figure 7).DiscussionThe initial screen 1480666 for protein variants to identify expressible constructs and to determine the optimal expression host for a given protein is the most time consuming process in a protein production pipeline using eukaryotic expression systems. To address this, we have successfully established a single expression vector for several eukaryotic hosts that allows direct analysis in transient gene expression (TGE), baculoviral expression (BEVS) and stable genomic expression methods (RMCE) in mammalian and insect cell lines. The versatile pFlp-Bac-to-Mam expression vectors allow a multiparallel approach comprising fast screening of expressible constructs without the need for recloning in the above-mentionedFigure 6. Expression profile of ECD mTLR2 in BEVS. Westen Blot analysis of the culture supernatant and intracellular fractions of Sf21 infected with recombinant pFlpBtM-II derived baculovirus producing ECD-mTLR2. Significant amounts of recombinant protein accumulate intracellularly as insoluble material due to compromised folding and secretion capability of virus infected cells. (SN = supernatant, S = intracellular soluble fraction, IS = intracellular insoluble fraction). doi:10.1371/journal.pone.0068674.gexpression systems. We implemented for the first time a combination between the potent RMCE system for fast generation of stable producer cell lines in 8 weeks, the well-known Tn7transposase based generation of recombinant bacmids for baculoviral expression in insect cells and transient transfection in EBNA1-expressing mammalian HEK293-6E cells. Since pFlpBtM can be used for both, fast transient and stable genomic expression in mammalian cells as well as a donor vector for the generation of recombinant bacmids it accelerates the initial screening for expressible constructs and the most suitable host for any given protein (Figure 8). In a comparative test expression with model proteins of three different protein classes, including a secretory scFv-Fc, the ECD of murine Toll like receptor 2 and the intracellular protein mCherry, the different expression strategies and hosts were evaluated. Each protein showed different expression characteristics in the tested hosts. Thereby it was possible to determine the optimal expressions strategy for each model protein. The intracellular yield of mCherry varied within one log scale between 8 mg/L in stable expression in the RMCE-CHO cell line and 52 mg/L in transient expression both in the BEVS and HEK293-6E system. Thus, the stable expression in RMCE based cell lines has to be considered as less favourable for the intracellular expression of the mCherry protein compared to expression with higher copy number in viral and plasmid-based transient systems. Parallel TGE of the scFv-Fc model protein in HEK293-6E was performed to benchmark the expression capability of pFlpBtM-II compared to the conventional pCMV vector and pTT5 which has been the dedicated expression plasmid for this cell line. Due to its multiple genetic elements pFlpBtM-II-scFv-Fc is 40 larger than pTT5-scFv-Fc and 30 larger compared to pCMV-scFv-Fc. Despite the resulting noteworthy decrease of the gene dose, TGE in HEK293-6E using pFlpBtM-I.MTLR2 in the BEVS. However, capturing ECD-mTLR2 from cell culture supernatants of the CHO producer cell lines provided a higher purity with less contamination compared to the expression in BEVS, where a significant contamination by host cell proteins is observed due to cell lysis (Figure 7).DiscussionThe initial screen 1480666 for protein variants to identify expressible constructs and to determine the optimal expression host for a given protein is the most time consuming process in a protein production pipeline using eukaryotic expression systems. To address this, we have successfully established a single expression vector for several eukaryotic hosts that allows direct analysis in transient gene expression (TGE), baculoviral expression (BEVS) and stable genomic expression methods (RMCE) in mammalian and insect cell lines. The versatile pFlp-Bac-to-Mam expression vectors allow a multiparallel approach comprising fast screening of expressible constructs without the need for recloning in the above-mentionedFigure 6. Expression profile of ECD mTLR2 in BEVS. Westen Blot analysis of the culture supernatant and intracellular fractions of Sf21 infected with recombinant pFlpBtM-II derived baculovirus producing ECD-mTLR2. Significant amounts of recombinant protein accumulate intracellularly as insoluble material due to compromised folding and secretion capability of virus infected cells. (SN = supernatant, S = intracellular soluble fraction, IS = intracellular insoluble fraction). doi:10.1371/journal.pone.0068674.gexpression systems. We implemented for the first time a combination between the potent RMCE system for fast generation of stable producer cell lines in 8 weeks, the well-known Tn7transposase based generation of recombinant bacmids for baculoviral expression in insect cells and transient transfection in EBNA1-expressing mammalian HEK293-6E cells. Since pFlpBtM can be used for both, fast transient and stable genomic expression in mammalian cells as well as a donor vector for the generation of recombinant bacmids it accelerates the initial screening for expressible constructs and the most suitable host for any given protein (Figure 8). In a comparative test expression with model proteins of three different protein classes, including a secretory scFv-Fc, the ECD of murine Toll like receptor 2 and the intracellular protein mCherry, the different expression strategies and hosts were evaluated. Each protein showed different expression characteristics in the tested hosts. Thereby it was possible to determine the optimal expressions strategy for each model protein. The intracellular yield of mCherry varied within one log scale between 8 mg/L in stable expression in the RMCE-CHO cell line and 52 mg/L in transient expression both in the BEVS and HEK293-6E system. Thus, the stable expression in RMCE based cell lines has to be considered as less favourable for the intracellular expression of the mCherry protein compared to expression with higher copy number in viral and plasmid-based transient systems. Parallel TGE of the scFv-Fc model protein in HEK293-6E was performed to benchmark the expression capability of pFlpBtM-II compared to the conventional pCMV vector and pTT5 which has been the dedicated expression plasmid for this cell line. Due to its multiple genetic elements pFlpBtM-II-scFv-Fc is 40 larger than pTT5-scFv-Fc and 30 larger compared to pCMV-scFv-Fc. Despite the resulting noteworthy decrease of the gene dose, TGE in HEK293-6E using pFlpBtM-I.
