Protein O GlcNAc transferase OGT is the sole enzyme to catalyse the attachment of beta N acetylglucosamine to the hydroxyl group of serine and threonine residues of intracellular proteins. N acetylglucosamination is an abundant dynamic post translational modification that shows extensive crosstalk with protein phosphorylation. Modified proteins are involved in a number of important cell processes and the disruption of homeostasis has been reported to be associated with many human diseases. Considering its biological importance, OGT inhibitors are a convenient tool for understanding the mechanisms of modification, discovering its unknown functions and validating OGT as a therapeutic target. The efficient production of functional proteins in heterologous host systems plays a crucial role in modern biotechnology. In the master s thesis, we focused on the optimization of OGT expression and purification. Our aim was to isolate an active and chromatographically pure protein of optimal concentration using the E. coli expression system. In the first part of the task we screened the expression at different inducer concentrations in three bacterial strains NiCo21DE3, Rosetta gami2DE3 pLysS and SHuffle T7 Express lysY. To improve protein expression, NiCo21DE3 and SHuffle T7 Express lysY expression systems were transformed with the plasmid pRARE2 harboring seven rare codon tRNAs. Analysis of cell lysates by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that OGT had the highest expression levels in the bacterial strain NiCo21DE3 transformed with pRARE2 and in the presence of 0.2 mM inducer. The second part represented a multistep purification of the protein obtained by optimized scale up expression. We probed the effects of the cell lysis methods on the correct protein structure, the removal of native E. coli proteins and techniques for concentrating proteins to achieve the desired amount and purity. We discovered that the purification by immobilized metal affinity chromatography followed by size exclusion chromatography led to the recombinant product of highest purity. Prior to that, bacterial cells were lysed by sonication. The recombinant enzyme was concentrated by ultrafiltration. All buffers were supplemented with 0.1 percent Tween 20. Finally, the concentration of protein was determined spectrophotometrically, and its purity was confirmed by polyacrilamide gel visualization. Enzyme s activity was assessed by a direct fluorescent activity assay.
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