- Poster presentation
- Open Access
INVect - a novel polycationic reagent for transient transfection of mammalian cells
© Püngel et al.; licensee BioMed Central Ltd. 2013
- Published: 4 December 2013
- Transfection Efficiency
- Transient Transfection
- Recombinant Protein Production
- Post Transfection
- Immobilize Metal Affinity Chromatography
For rapid recombinant protein production in small to medium size volumes, transient transfection of mammalian cells is still the method of choice in biotechnology . However, due to the high costs of commercially available lipofectamines or polycationic transfection reagents such as polyethylenimine (PEI), the most widely used transfection reagents available present a substantial economic bottleneck. While these reagents produce seemingly high transient transfection rates , there is still a strong desire for transfection reagents providing both secure and easy handling and higher recombinant protein production. As part of our commitment to excellence, InVivo BioTech Services initiated a joint venture with emp Biotech and developed a novel polycationic reagent, named INVect, for transient transfection and recombinant protein production in mammalian cells.
Mammalian cells were cultured in CD-ACF media using shake flasks and standard culture conditions. Cells were transfected with 10 μg per mL of a GOI harboring plasmid at a cell density of 5 × 106 cells per mL in FreeStyle™ Medium (Life Technologies) with INVect to DNA ratio of 6:1 (w/w) and PEI to DNA ration of 2:1 (w/w). Cultures were supplemented with same volume Protein Expression Medium (Life Technologies) 2 hours post transfection. GFP and SEAP expression took place in 8 mL culture volume in 50 mL bioreactor tubes. Expression of other reporter proteins were performed in 150 mL culture volume in 500 mL shake flasks. Transfection efficiency was determined 24 hours post transfection by counting green fluorescent positive cells using a FACSCalibur (Becton, Dickinson and Company). SEAP expression was determined in cell culture supernatant on day 6 post transfection by a photometric pNPP turn-over assay. Quantification of IgG was performed by protein G affinity chromatography on day 6 post transfection. Thrombomodulin concentration was calculated from cell culture supernatant on day 6 post transfection by IMUBIND® Thrombomodulin ELISA Kit (american diagnostica). His-tagged recombinant protein was purified on day 6 post transfection by TALON® immobilized metal affinity chromatography system.
INVect successfully delivers genes to HEK293-F, CHO-S and CAP-T cells as shown in a SEAP expression system (Figure 1C). Post-transfection cell productivity was determined under TGE manufacturing conditions. Thrombomodulin (60 kDa) (Figure 1D), an IgG (144 kDa) (Figure 1E) and a HIS-tagged Protein of Interest (~40 kDa) (Figure 1F) were transiently expressed using INVect as transfection reagent and conventional 25 kDa PEI as control. Cells were transfected with a gene of interest harboring plasmid, with product concentration being measured on day 6 post transfection. The use of INVect provided a minimum 2-fold increase in protein production over PEI (25 kDa) based transfection.
INVect is a novel polycationic transfection reagent which demonstrates low cell toxicity for transient transfection of mammalian cells and delivers extremely high transfection efficiencies of up to 90%, 24 h post transfection. The use of INVect for transfection under TGE conditions leads to exceptionally high levels of protein expression and outperforms 25 kDa linear PEI by 2-fold. INVect can be used effectively with all common cell lines and is especially suited for HEK293-F and CAP-T cells.
- Geisse S: Reflections on more than 10 years of TGE approaches. Protein Expr Purif. 2009, 64: 99-107.View ArticlePubMedGoogle Scholar
- Fischer S, Charara N, Gerber A, Wölfel J, Schiedner G, Voedisch B, Geisse S: Transient recombinant protein expression in a human amniocyte cell line: the CAP-T® cell system. Biotechnol Bioeng. 2012, 109: 2250-2261.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.