Protein purification procedure normally has to be custom-tailored for each protein, as it depends on protein properties, as well as the purpose of protein production. Optimization of the procedure can take a long time and significant amount of the sample, hence the protein purification was (and often still is) considered as the bottleneck of protein research. Affinity tags are proven to be tremendously effective tools for a wide variety of applications, primarily in shortened protein purification protocols, but also e.g. in immobilization of proteins for display on a surface, in detection and quantification of target proteins, in analysis of protein-ligand and protein-protein interactions. Today, IMAC (Immobilized-metal affinity chromatography) is used routinely for purification of proteins for various purposes, and it is generally thought that small size and neutral charge of His-tag don’t influence structure and function of the protein. However, more and more reports are published stating negative effect of His-tag on protein’s oligomerization state, structure, dynamics, function and activity. We have constructed two variants of adenylosuccinate synthetase (AdSS) from Helicobacter pylori – with His6-tag on N- or C-terminus. Both variants were purified (by IMAC and size-exclusion chromatography), characterized (protein stability, enzyme kinetics) and their properties compared together with wild-type variant of the enzyme. N-His-AdSS and C-His-AdSS were also crystallized and their 3D-structures determined. All our results combined indicate diminishing effect of His6-tag only on N-terminus of H. pylori AdSS on the enzyme’s properties.