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Recombinant expression, purification and characterisation of the surface immunoreceptor CD47 (integrin associated protein)
Kuchyňová, Sarah ; Konvalinka, Jan (advisor) ; Vaněk, Ondřej (referee)
The human immunoreceptor CD47, also designated <Integrin Associated Protein= is a novel therapeutic target for cancer treatment. When bound to one of its ligands, a protein found on the surface of immune cells, SIRP, the phagocytosis of the CD47 expressing cell is inhibited. This interaction is used by malignant cells to escape detection by the immune system, thus making CD47 a promising candidate molecule for cancer immunotherapy. The literary overview focuses on the characterisation of CD47 in terms of its structure, localization, and function. Additionally, this sections thoroughly delves into the aforementioned interaction with SIRP as well as existing mechanisms for the targeting of the CD47- SIRP axis. The following experimental section centers around the preparation of the plazmid pTT28_hCD47. The process by which the gene for the extracellular portion of human CD47 was inserted into the linearized pTT28 vector is noted and its transfection into a mammalian expression system to produce the protein. Moreover, the section divulges into the purification of the recombinant protein by affinity purification using Ni-NTA agarose as well as gel permeation chromatography, resulting in the isolation of CD47 proteins differing in glycosylation. Their functionality was validated via SPR, in a study...
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