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Directed evolution of enzymes on microfluidic chip
Kohúteková, Táňa ; Chmelíková, Larisa (referee) ; Prokop,, Zbyněk (advisor)
Tato práce se zaměřuje na řízenou evoluci enzymů pomocí fluorescenčně řízeného kapénkového třídení (ang. zkratka FADS) na mikrofluidním čipu. Byl sestaven nový třídicí systém, který byl validován pro vysoce výkonnou optickou detekci mikrofluidních kapének spojenou s dielektrickým tříděním. Systém byl testován pomocí dvou různých (DhaA31, ancHLD-RLuc) populací haloalkan dehalogenas (HLD) exprimovaných v buňkách Escherichia coli a jedné populace prázdného vektoru pIDR9 v poměru 5:45:50, což imituje proteinovou knihovnu. Naměřená a analyzovaná data z experimentu ukazují, že intenzity kapek ve validačním experimentu byly zaznamenány v stejném poměru jako teoretický poměr. Poté byla knihovna HLD mutantů s inzercemi a delecemi tříděna pomocí FADS, za účelem získaní 1 % nejaktivnějších variant. Program pro analýzu dat pro FADS byl programován v prostředí LabVIEW s využítím jazyka Python, s funkcemi pro počáteční analýzu, predikci prahového napětí, validaci a grafickou interpretaci dat a konečnou zprávu o experimentu.
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Engineering and selection of protein binders recognising medically important cytokines
Huličiak, Maroš
Protein engineering attracts more attention as a powerful tool of biotechnology and medicine. Small, engineered proteins derived from protein molecules of stable fold, the so called scaffolds, are potential replacements of supplements of more widely used antibodies. In this thesis, I introduce utilization of two scaffold molecules designed in our laboratory for development of stable and specific protein binders of high affinity. This thesis discusses the development of binders interacting with medically important human cytokines and their cellular receptors, interleukin-10, interleukin-28 receptor, and interleukin-9 receptor alpha. Recombinant cytokine and receptor proteins were expressed in eukaryotic cells in high yields and quality and served as molecular targets for selections using various display methods of directed evolution. We demonstrated that application of ribosome and yeast display methods or their unconventional combination in a newly developed integrated pipeline leads to successful generation of high affinity and specificity binders based on newly designed protein scaffolds called 57aBi and 57bBi.
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Engineering and selection of protein binders recognising medically important cytokines
Huličiak, Maroš ; Schneider, Bohdan (advisor) ; Pichová, Iva (referee) ; Kukačka, Zdeněk (referee)
Protein engineering attracts more attention as a powerful tool of biotechnology and medicine. Small, engineered proteins derived from protein molecules of stable fold, the so called scaffolds, are potential replacements of supplements of more widely used antibodies. In this thesis, I introduce utilization of two scaffold molecules designed in our laboratory for development of stable and specific protein binders of high affinity. This thesis discusses the development of binders interacting with medically important human cytokines and their cellular receptors, interleukin-10, interleukin-28 receptor, and interleukin-9 receptor alpha. Recombinant cytokine and receptor proteins were expressed in eukaryotic cells in high yields and quality and served as molecular targets for selections using various display methods of directed evolution. We demonstrated that application of ribosome and yeast display methods or their unconventional combination in a newly developed integrated pipeline leads to successful generation of high affinity and specificity binders based on newly designed protein scaffolds called 57aBi and 57bBi.
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Modulation of interactions of cytokines and their receptors
Kolářová, Lucie ; Schneider, Bohdan (advisor) ; Rozbeský, Daniel (referee) ; Osička, Radim (referee)
Protein-protein interactions and interactions with other molecules including DNA and RNA, play an important role in a range of biological activities and processes in all living cells. Understanding of protein-protein interactions, new approaches, and tools for their modulations are valuable for medicine, biotechnology, and drug development. We used the interleukin-10 family of cytokines as a model system for our research of biological interactions and modulation of their functions. A key prerequisite to study biological processes and a detailed understanding of biomolecular interactions is a recombinant protein that is stable under a broad range of conditions. Recombinant protein expression in sufficient yield and quality is often a challenging task. Therefore, we aimed at developing new approaches in protein design and production. In the first part of our study, we modified IL-24, a member of the IL-10 family to increase its expression and stability. We demonstrated that protein engineering is a powerful tool in research of difficult protein targets. In the second part of our study, we adopted new approaches in designing new protein scaffolds suitable for use in the ribosome and yeast display techniques. Protein scaffolds have become promising alternatives to antibodies in protein drug...
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Directed evolution of mouse polyomavirus
Váňová, Jana ; Španielová, Hana (advisor) ; Mašek, Tomáš (referee)
The method of directed evolution represents a new approach to generate proteins with new or altered properties. The principle of directed evolution is random mutagenesis of the coding sequence for a protein of our interest followed by selection of generated mutants for the desired property. The aim of this pilot study was to investigate the possibility of utilization of directed evolution for alteration of mouse polyomavirus original tropism and virus retargeting to a model prostate cancer cell line. To generate randomly mutated gene encoding the major capsid protein of mouse polyomavirus, which is responsible for the interaction of the virus with cellular receptor for viral cell entry, error-prone PCR and DNA shuffling methods were used. Production of viruses composed of mutant major capsid protein was ensured by Cre/loxP site-specific recombination. The thesis also dealt with the design and characterization of the system for viral mutant selection. It was found that the prostate cancer cell lines markedly vary in their ability to bind and internalize particles derived from mouse polyomavirus. This knowledge can be used for the preparation of virus-like particles for prostate cancer diagnostics in the future. The study demonstrated that the method of directed evolution can be used for production...
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Retargeting of viral particles for directed cargo delivery into cells
Váňová, Jana ; Španielová, Hana (advisor) ; Kuthan, Martin (referee)
Due to their structure and ability to enter cells where they release their content, viral particles represent an attractive tool to deliver cargo to a cell. For therapeutic usage of viruses it is necessary to ensure the specific and highly efficient entry to target cells. This thesis offers an overview of methods used for virus retargeting with the intention to evaluate the success of retargeting in terms of specificity and efficiency of designed viral particles when entering a cell. On the basis of published data and considering the mechanisms of viral infection, the thesis demonstrates the difficulty to prevent the nonspecific viral particles from entering the cell and concludes that reaching total specificity is apparently impossible. Despite these small limitations, viral nanoparticles are a revolutionary therapeutic tool for delivering cargo to the cell and it is necessary to exploit their potential.
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