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Study of modified amino acid incorporation into cytochrome b5
Koberová, Monika ; Hodek, Petr (advisor) ; Pavlásková, Kateřina (referee)
A cytochrome b5 (cyt b5) can influence cytochrome P450 (CYP)-dependent reactions. In consequence of these reactions cytochrome b5 can participate in substance activation (for example drugs, carcinogens) or it can influence proportions of formed metabolites. A mechanism of cyt b5 action has not been fully explained yet. Elucidation of protein-protein interactions in monooxygenase system could explain of the mechanism of cyt b5 action. To study these interactions by using cross-linking techniques is necessary to prepare photolabile cyt b5, which after photoactivation generated higly reactive intermediates which can create a complex with nearby components of the monoogynesase system. This thesis describes how was developed the method for the production of recombinant cyt b5 with modified amino acids. Cyt b5 was expressed in a bacterial strain E. coli BL-21 (DE3) Gold. Before the expression induction, cells were transformed into the limiting medium (DMEM-LM) which did not contain L-leucine and L-methionine. The limiting medium was supplemented by deuterated amino acid d3-methyl-L-methionine and D,L-Leucine. Expressed cyt b5 was isolated and incorporation of d3-methyl-L-methionene has been verified by mass spectrometry. Cyt b5 was obtained mainly as the apoprotein (apo-cyt b5). That is why in this...

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