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Posttranslational modifications in soluble recombinant therapeutical proteins secreted by lower aukaryotes: structure and function
Kumar, Vinay ; Bezouška, Karel (advisor) ; Šedo, Aleksi (referee) ; Grubhoffer, Libor (referee)
Virtat, Kunnr M.Sc Aint of the stud1, Aim of the studv The aim of this study was to understand the molecular mechanisnrs contributing to the production of soluble leukocyte receptors in the eukaryotic expression systenrs including the proper posttranslational modifications such as disulfide bond fornration and glycosylation. In order to achieve this goal, the following specific aims have been adopted for the study: l, t. To develop new methodologies for rapid and convenient assessnrent ofthe disulfide L I boltds in complex eukaryotic proteins using SDS polyacrylarride electrophoresis under nonreducing conditions, arrd nrass spectrometry. 2. To establish elements that are critical for the stability of soluble CD69 receptors expressed in the bacterial expression systenr 3. To develop methods for the production of glycosylated soluble CD69 proteins in lower eukaryotes (P i c h i a pa s to ri s) 4. To purify the proteins in sufficient quantities for biochemical and inrnrunological studies 5. To investigate, how disulfide bonding and glycosylation influeuces the stability of soluble CD69 receptors 6. To look at the carbohydrate binding activities ofthe produced proteins. 7. To evaluate the in vivo propefties of the produced proteins including their circulation half lifes in the blood of experiental animals, and...

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