|
|
The influence of S159A mutation on the oligomeric state of human NK cell receptor NKR-P1A
Hausleitner, Filip ; Vaněk, Ondřej (advisor) ; Kožíšek, Milan (referee)
Natural killer cells (NK cells) are lymphocytes that possess cytotoxic activity against tumour or virally infected cells independent of preceding antigen sensitisation. To kill such cells, they utilise their activating and inhibitory surface receptors that interact with target cell surface molecules. The immune response carried by NK cells depends on the balance of both activating and inhibitory signals. Human NK cell surface receptor NKR-P1A belongs to the structural family of C-type lectin-like receptors. This receptor interacts with its ligand LLT1, which belongs to the same protein family, with low affinity and high specificity. The NKR-P1A:LLT1 complex formed between NK cell and its target cell inhibits NK cell cytotoxicity, and hence is a part of the regulation of immune response. This thesis studied the effect of S159A mutation on the stoichiometric state of soluble human NKR-P1A ectodomain in solution. Therefore, a mutant form of NKR-P1A G90-S225 S159A ectodomain was successfully produced in stably transfected human embryonic kidney cells 293 (HEK293S GnTI" ). This construct was purified by affinity and size-exclusion chromatography, and analysed by SDS-PAGE and analytical ultracentrifugation. Our results show that the preclusion of N-linked glycosylation in the position 157 promotes the...
|
|
|
Structural characterization of intracellular form of mice protein Nkr-p1a
Vaňková, Pavla ; Novák, Petr (advisor) ; Kolenko, Petr (referee)
NK cells are a component of innate immunity system, which is derived from lymphoid progenitor. By a sophisticated receptor repertoire, which is expressed on their surface, they provide a surveillance against pathogenic, virus infected or tumour cells. Simultaneously they produce cytokines, thereby are involved in adaptive immune response. This work is focused on the study of structure of mice soluble mNkr-p1a isoform. Recently this short isoform was identified at the transcriptional level by a member of our laboratory and it is designated as isoform 2. The aim was to produce mNkr-p1a iso2 protein in the prokaryotic expression system and to perform its renaturation and purification in vitro. In the next phase of work, the obtained product was analyzed by the mass spectrometry methods. Recieved results made us think about that our protein is in unfolded state. This assumption was refuted by following biophysical methods, nuclear magnetic resonance, circular dichroism and dynamic light scattering measurement. Keywords: NK cells Receptor mNkr - p1a Short isoform mNkr - p1a iso2 Alternative splicing Protein biosynthesis Recombinant protein production Protein purification Mass spectrometry Disulfide bond Chemical cross-linking NMR, CD, DLS 5
|
guest ::
