Recombinant proteins belong to considerable biofarmaceutics products used in biomedical research and in the treatment of human disease. Recombinant protines can be produced by stable transfection in big amount or by faster transient transfection with smaller amounts. To provide regular biological activity, it is necessary for the protein to be properly folded and post-translationally modified. As these modifications can be accurately performed only in mammalian cells, they have become the major host for complex r-protein expression. In this thesis is described transient transfection HEK 293 EBNA1 cells with linear polyethylenimines. These cells has been adapted to suspension cultivation in serum free medium. The cells were transfected with pcDNA3.1, pCI, pEBSV1, pCEP4, pEAK8 a pcDNA5/FRT/TO plasmids, everyone contained repoter gene SEAP. Concentration of SEAP in cell culture supernatants were determined in order to compare efficiencies of individual transfections. DNA:PEI ratio was another factor which was optimised and two different PEIs were compared. Highest achieved expresion was 50 mg per litre with transfection in 24 well plate when DNA:PEI ratio was 1:5. Comparison of six different plasmids give the bigest expresion pCEP4/SEAP, in well plate as well as in scaled up system.

Natural killer (NK) cells are an intensively studied part of immune system, possessing unique ability to recognize and induce death of tumor and virus-infected cells without prior antigen sensitization. Their function is regulated by a fine balance of signals induced by multiple activating and inhibitory cell surface receptors and their interaction with the ligands present on the target cell. Recent research in their C-type lectin-like receptors repertoire has shown that ligands of some of these previously orphan receptors lie within their own family, describing a lectin-lectin interaction. This is the case of human inhibitory receptor NKRP1 (gene KLRB1) and its ligand LLT1 (gene CLEC2D). Previous studies have shown that overproduction of LLT1 in cancer cells or lower production of NKRP1 in NK cells is connected to cancerous manifestations. This master's thesis shows a successful production of the extracellular part of LLT1 utilizing a mammalian expression system based on transient transfection of modified human embryonic kidney (HEK) cell lines. It was found that the five cystein residues contained within the lectin domain of LLT1 tend to cause misfolding and formation of aggregates. Stabilization of the domain was achieved by restoration of the sixth cystein residue at the evolutionary conserved...

Transient transfection of mammalian cell lines is an effective approach for recombinant protein production, which can provide milligrams to grams of proteins in two weeks from cloning of the corresponding cDNA. Native glycosylated proteins prepared via this approach can be used for various purposes in molecular biology, immunology or pharmaceutical industry, i.e. initial phase of pre-clinical therapeutic protein research. One of the most used mammalian host cell lines is the human embryonic kidney cell line, that can be easily cultivated and chemically transfected. The amount of proteins produced by transiently transfected human embryonic kidney cells can be enhanced by a whole range of factors, i.e. co-expression or direct addition of acidic fibroblast growth factor to the culture medium, co-expression of cell cycle regulating proteins or anti-apoptotic proteins. Expression plasmid pTW5 was prepared and further modified by gene insertion of aFGF, cell cycle regulator p18, p21 or p27 (cyclin-dependent kinase inhibitors) or apoptosis inhibitor bcl-2 or bcl-x. These plasmids were then used for optimization of HEK293T cell line expression system. The impact of every single regulator and their combinations, including hitherto undescribed effect of combination of cell cycle regulator and anti-apoptotic...

Actuellement le polyéthylènimine (PEI) est l’agent de transfection transitoire le plus utilisé par l’industrie pharmaceutique pour la production de protéines recombinantes à grande échelle par les cellules de mammifères. Il permet la condensation de l’ADN plasmidique (ADNp) en formant spontanément des nanoparticules positives appelées polyplexes, lui procurant la possibilité de s’attacher sur la membrane cellulaire afin d’être internalisé, ainsi qu’une protection face aux nucléases intracellulaires. Cependant, alors que les polyplexes s’attachent sur la quasi-totalité des cellules seulement 5 à 10 % de l’ADNp internalisé atteint leur noyau, ce qui indique que la majorité des polyplexes ne participent pas à l’expression du transgène. Ceci contraste avec l’efficacité des vecteurs viraux où une seule particule virale par cellule peut être suffisante. Les virus ont évolués afin d’exploiter les voies d’internalisation et de routage cellulaire pour exprimer efficacement leur matériel génétique. Nous avons donc supposé que l’exploitation des voies d’internalisation et de routage cellulaire d’un récepteur pourrait, de façon similaire à plusieurs virus, permettre d’optimiser le processus de transfection en réduisant les quantités d’ADNp et d’agent de transfection nécessaires. Une alternative au PEI pour transfecter les cellules de mammifèreest l’utilisation de protéines possédant un domaine de liaison à l’ADNp. Toutefois, leur utilisation reste marginale à cause de la grande quantité requise pour atteindre l’expression du transgène. Dans cette étude, nous avons utilisé le système E/Kcoil afin de cibler un récepteur membranaire dans le but de délivrer l’ADNp dans des cellules de mammifères. Le Ecoil et le Kcoil sont des heptapeptides répétés qui peuvent interagir ensemble avec une grande affinité et spécificité afin de former des structures coiled-coil. Nous avons fusionné le Ecoil avec des protéines capables d’interagir avec l’ADNp et le Kcoil avec un récepteur membranaire que nous avons surexprimé dans les cellules HEK293 de manière stable. Nous avons découvert que la réduction de la sulfatation de la surface cellulaire permettait l’attachement ciblé sur les cellules par l’intermédiaire du système E/Kcoil. Nous démontrons dans cette étude comment utiliser le système E/Kcoil et une protéine interagissant avec l’ADNp pour délivrer un transgène de manière ciblée. Cette nouvelle méthode de transfection permet de réduire les quantités de protéines nécessaires pour l’expression du transgène. / Pharmaceutical industry often employs polyethylenimine (PEI) for large scale protein production processes by transient transfection of mammalian cells. PEI condenses plasmid DNA (pDNA) by spontaneously forming positive nanoparticles known as polyplexes. Condensed pDNA is favoured for cell surface binding, internalization and protection from intracellular nucleases. While most of the cells efficiently uptake polyplexes, only 5 to 10% of captured pDNA reaches the nucleus for transgene expression. This suggests that polyplexes are hampered in their ability to route and to translocate to the nucleus necessitating large amounts of polyplexes to achieve high expression levels. By contrast, many viruses can efficiently transduce cells with only one or a few viral genome copies. Viruses have evolved to exploit cellular internalization and routing properties to express their own genetic material. We hypothesized that less pDNA would be used in an optimized transfection process if we exploited the internalization and routing properties that viruses use. DNA binding proteins could be used as an alternative to PEI to transfect mammalian cells. However, their usage is marginal due to the large protein quantities required to bind pDNA for transgene expression. If less pDNA is used less binding protein is needed. In this study, we used the E/Kcoil system to target a membrane receptor to deliver pDNA in mammalian cells. The Ecoil and Kcoil are two repeated heptapeptides which interact with a high affinity and specificity to form coiled-coil structures. We fused the Ecoil with a recombinant pDNA-binding protein. The Kcoil was fused to a stably-expressed membrane receptor in HEK293 cells. We discovered that low sulfation of the cell surface reduced non-specific binding of the pDNA:protein complex and permitted targeted binding via the E/Kcoil interaction. We demonstrate how to use recombinant pDNA-binding protein and the E/Kcoil system for targeted transgene delivery. This newly developed system provides a new transfection method, with reduced pDNA-binding protein quantities needed to achieve transgene expression.