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Preparation of expression constructs of phosducine binding partners.
Koláčková, Kateřina ; Obšil, Tomáš (advisor) ; Vaněk, Ondřej (referee)
Key words: SUG1, phosducin, G protein signaling, expression, pGEX-4T-1, pST39 Phosducin is an acidic protein found in cytosol and is involved in G protein signaling within rods of photoreceptors. Phosducin also influences stress-dependent hypertension. One of phosducin's important binding partners is SUG1, a 26S proteasome subunit, which belongs to a family of proteins that have an ATPase activity. This bachelor thesis takes part in a broader research, which tries to clarify physiological functions of the interaction of SUG1 with phosducin. Main goal of this study was to prepare constructs of SUG1 (human isoform 1) suitable for the recombinant protein production in an expression system E. coli BL21 (Rosetta). Gene that encodes SUG1 was inserted into two different plasmid DNAs that allow expression of SUG1 as two different fusion proteins in a prokaryotic expression system. Nucleotide sequences of these constructs were verified using sequencing. Next, expression tests revealed sufficient production of both recombinant fusion proteins. The main result of this bachelor thesis is the successful expression of human SUG1 in E. coli BL21 (Rosetta) cells. This result will enable the development of the purification protocol for the large scale expression of both recombinant fusion proteins as well as the...
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Preparation and characterization of binding partners of phosducin.
Kylarová, Salome ; Obšil, Tomáš (advisor) ; Teisinger, Jan (referee)
AABBSSTTRRAACCTT Phosducin (Pdc) is a highly conserved acidic phosphoprotein, which plays an important role in the regulation of G-protein signalization in intact retina. It binds to Gβγ dimer of heterotrimeric G-protein transducin thereby decreases the pool of available transducin resulting in modulation of signal. Function of phosducin is negatively regulated by its phosphorylation followed by interaction with the 14-3-3 protein. Besides this established way of regulation, we were interested in other putative interaction partners of phosducin, like SUG1 and CRX. SUG1 is a subunit of 26S proteasome with a large scale of biological functions, especially a degradation of many transription factors. Its role in regulation of phosducin is still unclear, but is probably involved in targeting of phosducin to 26S proteasome for its degradation. Subsequently, we prepared four different expression constructs of full-length protein in order to find the best expression and purification strategy. These results suggest that all purified fusion proteins of SUG1 form stable and soluble high molecular weight oligomers. This behaviour was confirmed by dynamic light scattering and analytical ultracentrifugation measurements. In addition, this observation is consistent with previous studies of its bacterial counterpart, PAN...
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Preparation of expression constructs of phosducine binding partners.
Koláčková, Kateřina ; Obšil, Tomáš (advisor) ; Vaněk, Ondřej (referee)
Key words: SUG1, phosducin, G protein signaling, expression, pGEX-4T-1, pST39 Phosducin is an acidic protein found in cytosol and is involved in G protein signaling within rods of photoreceptors. Phosducin also influences stress-dependent hypertension. One of phosducin's important binding partners is SUG1, a 26S proteasome subunit, which belongs to a family of proteins that have an ATPase activity. This bachelor thesis takes part in a broader research, which tries to clarify physiological functions of the interaction of SUG1 with phosducin. Main goal of this study was to prepare constructs of SUG1 (human isoform 1) suitable for the recombinant protein production in an expression system E. coli BL21 (Rosetta). Gene that encodes SUG1 was inserted into two different plasmid DNAs that allow expression of SUG1 as two different fusion proteins in a prokaryotic expression system. Nucleotide sequences of these constructs were verified using sequencing. Next, expression tests revealed sufficient production of both recombinant fusion proteins. The main result of this bachelor thesis is the successful expression of human SUG1 in E. coli BL21 (Rosetta) cells. This result will enable the development of the purification protocol for the large scale expression of both recombinant fusion proteins as well as the...
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