| Abstract [eng] |
..........SUMMARY The aim of this thesis was to clone the SUP35NM gene into a bacterial expression vector and to optimize the synthesis of the Sup35 protein in E. coli expression strains. Methods of molecular cloning, optimization of recombinant protein synthesis, transformation using a pulsating electric field, cultivation of microorganisms, and visualization of protein profiles were performed during the research. The SUP35NM-GFP gene was successfully cloned into the pET-21c (+) expression vector after successful bioinformatics analysis, restriction endonuclease selection, DNA isolation, and ligation of DNA fragments. The pET-21c (+) - SUP35NM-GFP construct was introduced into E. coli BL21 (DE3) and E. coli Rosetta (DE3) expression strains. Synthesis and optimization of the Sup35NM protein using these expression systems continued. Protein synthesis was not obtained. After the electrotransformation of E. coli BL21 (DE3) cells using the construct - pRSETB-8xHis-SUP35NM, the synthesis and its optimization of 8xHis-Sup35NM protein was performed, SDS-PAGE analysis showed that no protein synthesis took place. Two control expression systems were then selected: E. coli BL21 (DE3) StarTM pRSETB-8xHis-SUP35NM and E. coli BL21 (DE3) pET-21c (+)-GD25 After protein synthesis and optimization, NDS-PAGE gels showed that 8xHis-Sup35NM protein synthesis did not occur using the BL21 (DE3) StarTM pRSETB-8xHis-SUP35NM expression system although, Gd25 protein synthesis occurred using E. coli BL21 (DE3) pET-21c (+)-GD25 expression strain, NDS-PAGE gels show a 43 kDa protein that corresponds to the theoretical size of lipase Gd25. |
