ADP-ribosylation is a reversible post-translational modification in which adenosine-diphosphate-ribose (ADP-ribose) is transferred from nicotinamide-adenine-dinucleotide (NAD) to the target protein. The enzymes which transfer ADP-ribose onto target proteins are ADP-ribosyl-transferases, while the enzymes which reverse this modification are ADP-ribosyl hydrolases. ADP-ribosylation controls many important cellular processes in eukaryotes and prokaryotes. Bacteria of the genus Streptomyces, in particular S. coelicolor, are good model organisms for studying ADP-ribosylation in bacteria because they have genes for several enzyme types that might be included in ADP-ribosylation and de-ADP-ribosylation reactions. In addition, ADP-ribosylation regulates the production of antibiotics which are secondary metabolites produced by these bacteria. Several target proteins for ADP-ribosylation have been identified in S. coelicolor. In this research recombinant DNA technology has been used to clone the genes from five selected proteins from genome of S. coelicolor bacterium, which have been proven to be ADP-ribosylated. Genes are overexpressed, while recombinant proteins are purified by affinity chromatography. The testing was carried out on purified target proteins as substrates in reaction of ADP-ribosylation in vitro by using the enzyme ADP-ribosyl-transferase SCO5461 of which activity has been previously known, but its protein substrates have been unknown. ADP-ribosylation reaction in vitro has confirmed the ability of SCO5461 enzyme to modify itself and also demonstrated its ability to modify three selected target proteins. This work contributes to the research of ADP-ribosylation in streptomyces, which will provide better understanding and control of metabolic pathways of these important producers of numerous bioactive compounds.