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Expression and purification of protein containing photo-methionine - the use of photo-induced cross-linking to map protein-protein interaction
 Tichá, Andrea ; Ptáčková, Renata (advisor) ; Indra, Radek (referee)
Protein 14-3-3ζ is an important protein that plays a role in the regulation of cell survival and keeps cell from stress and possible apoptosis. A photo-activatable group can be incorporated into the protein to detect protein-protein interaction by photo induced crosslinking. The aim of this work was to create protein with incorporated photo-methionine in auxotrophic bacteria E. coli B834 and to optimize expression conditions. Photo-methionine is structurally very similar to natural methionine, so photo-methionine can be incorporated into the protein by host aminoacyl tRNA synthetase. It has been studied how the rate of incorporation varies depending on production time. After 3 or 6 hours, up to 70 % incorporation of photo-methionine was achieved compared to about 50 % after one hour.
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