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Recombinant preparation of isotopically labeled receptor rNKR-P1A for NMR studies
Čonka, Martin ; Bezouška, Karel (advisor) ; Novák, Petr (referee)
anglicky NK cells belong to the population of cells which are able to cytotoxic kill certain tumor and cells infected by viruses. This bachelor thesis focuses on the rat's NK cell receptor, especially rNKR-P1A. This protein belongs to the activating receptors and is able to activate the cytotoxic function of NK cells. Physiological ligand and three-dimensional structure of this protein has not been resolved yet. The aim of this work was optimalization of production rNKR-P1A in minimal medium and thereafter producing isotope-labeled proteins. Isotope- labeled proteins could contribute to solving the three-dimensional structure of this rat receptor by using NMR methods.
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Expression of the recombinant extracellular parts of leukocyte receptors AICL a NKR-P1CBALB
Čonka, Martin ; Novák, Petr (advisor) ; Cebecauer, Marek (referee)
v anglickém jazyce NK cells represent a population of lymphocytes which are able to kill certain tumor cells or virally infected cells. The subject of the diploma thesis is a mouse NK cell receptor mNKR- P1CBALB and a human leukocyte receptor hAICL. The mNKR-P1CBALB belongs to the activating receptors and is able to activate the cytotoxic functions of NK cells. The hAICL receptor is a ligand to the NKp80 which is an activating receptor of NK cells. Interaction between these two proteins leads to the activation of effector functions of NK cells as well. The aim of this work was the preparation of the recombinant extracellular parts of receptors mNKR-P1CBALB and hAICL, the optimalization of their in vitro refolding and the characterization of proteins using mass spectrometry. The proteins samples will be used for further structural study of the extracellular parts of these leukocytes receptors.
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Preparation of the soluble form of a mouse NKR-P1A protein for the NMR study
Skála, Kristián ; Chmelík, Josef (advisor) ; Adámková, Lyubina (referee)
Natural killer cells (NK cells) are a type of lymphocyte. According to their function they are defined as cytotoxic cells which cause cell death without prior sensitization. NKR-P1 is one of the families of NK surface receptors. This family belong to C-type lectin like with inhibitory or activatory function. In this work we concern of soluble form of mouse protein Nkr-p1a, that is isoform of activatory receptor Nkr-p1a. This receptor is expected to be intracellular due to lack of major part of its transmembrane domain. We focus on the optimization of Nkr-p1a production parameters. As production system we used bacterial strain E. coli BL21(DE3) Gold, in which the target protein is produced and subsequently isolated in the form of inclusion bodies. Obtained recombinant protein was refolded and purified. As purification step we used high-performance liquid chromatography. We optimized concentration of inductor of expression, production time and temperature. The objective is to set up protocol for preparation of isotopically labeled protein for nuclear magnetic resonance structure characterization. (in czech)
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Expression of the recombinant extracellular parts of leukocyte receptors AICL a NKR-P1CBALB
Čonka, Martin ; Novák, Petr (advisor) ; Cebecauer, Marek (referee)
v anglickém jazyce NK cells represent a population of lymphocytes which are able to kill certain tumor cells or virally infected cells. The subject of the diploma thesis is a mouse NK cell receptor mNKR- P1CBALB and a human leukocyte receptor hAICL. The mNKR-P1CBALB belongs to the activating receptors and is able to activate the cytotoxic functions of NK cells. The hAICL receptor is a ligand to the NKp80 which is an activating receptor of NK cells. Interaction between these two proteins leads to the activation of effector functions of NK cells as well. The aim of this work was the preparation of the recombinant extracellular parts of receptors mNKR-P1CBALB and hAICL, the optimalization of their in vitro refolding and the characterization of proteins using mass spectrometry. The proteins samples will be used for further structural study of the extracellular parts of these leukocytes receptors.
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Recombinant preparation of isotopically labeled receptor rNKR-P1A for NMR studies
Čonka, Martin ; Novák, Petr (referee) ; Bezouška, Karel (advisor)
anglicky NK cells belong to the population of cells which are able to cytotoxic kill certain tumor and cells infected by viruses. This bachelor thesis focuses on the rat's NK cell receptor, especially rNKR-P1A. This protein belongs to the activating receptors and is able to activate the cytotoxic function of NK cells. Physiological ligand and three-dimensional structure of this protein has not been resolved yet. The aim of this work was optimalization of production rNKR-P1A in minimal medium and thereafter producing isotope-labeled proteins. Isotope- labeled proteins could contribute to solving the three-dimensional structure of this rat receptor by using NMR methods.
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