In this master’s thesis we optimized isolation process of functional amiloid TasA protein, a major component of the Bacillus subtilis biofilm. Protein was isolated from Escherichia coli cells, which produce recombinant TasA using inductor of lac operon. TasA protein is produced as monomer or fibril. Fibril is the structure important for biofilm formation. TasA enables adhesion of cells to the surface and to themselves. Protein also contributes to mechanical stability of biofilm. During optimization of protein isolation, we cultured cells in different culture media with using different inductors of lac operon, which contributes to culture with higher cell density and simultaneously to higher protein production. Isolation was more effective using optimized induction culture medium, which contains MgSO4 and adding inductor isopropyl β- d-1-thiogalactopyranoside. Important role in cell growth has aeration of culture and proportion of inoculum. The best cultivation method was shaking 200 mL of culture in 500 mL Erlenmayer flask with baffles and 1 % inoculum. We also optimized sonication of E. coli suspension, considering produced recombinant TasA is an intracellular protein. Sonication for 1 L of culture was performed in 40 minutes using 30 seconds long sonication intervals and 15 micron amplitude of sonication strength. To gain purified TasA, we also optimized the process of protein purification using GST beads, TEV protease and Ni-NTA agarose beads. We determined the concentration of isolated protein using different analytical methods, UV-VIS spectrophotometry and SDS-PAGE electrophoresis. SDS-PAGE electrophoresis also enabled determining protein size and therefore we defined that isolated protein is a pure TasA protein.
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