Escherichia coli (E. coli) is an extremely versatile organism. It is a commensal bacterium that inhabits the gastrointestinal tract of vertebrates and is a crucial part of their microbiota. On the other hand, this bacterium is an important pathogenic organism that causes intestinal and extraintestinal infections. Amongst the very common extraintestinal pathogenic E. coli are uropathogenic E. coli (UPEC). UPEC are usually classified in B2 phylogenetic group and are responsible for 80 % of urinary tract infections (UTI). Most of UPEC strains have gene for Usp — uropathogenic specific protein. Usp is nuclease pyocins and colicins homolog and has DNase activity in vitro. At first it was anticipated that Usp is a bactericide, but later it was shown that it does not have bactericidal effect on bacteria, but a genotoxic effect on eukaryotic cells — therefor Usp is a genotoxin. Downstream of usp gene there are three genes for Imu proteins, which protect producing cells from Usp nuclease activity. Imu3 reversibly binds DNA and promotes genotoxic effect of Usp protein. The aim of this master’s thesis was to prepare Usp-GFP fusion recombinant protein with activity similar to the native Usp protein activity. Usp-GFP production was optimized. We grew E. coli strain BL21 (DE3) pLyseE transformed with pTHCG12U on 30 °C and induced Usp-GFP protein overexpression with 0.1 mM IPTG. Protein was then isolated with Ni-NTA affinity chromatography and additionally purified with FPLC. Activity was tested afterwards. Imu3 and Usp isolates were also prepared. We showed that Usp-GFP has DNase activity and effect on HUVEC cells biomass amount. Usp-GFP in vitro and in vivo activity is comparable to native Usp activity. Usp-GFP protein is a useful tool for further research of interaction between Usp and eukaryotic cells.