O-ClcNAc is an important post-translational modification of various proteins. The removal of O-GlcNAc is catalyzed by human O-GlcNAcase enzyme. The thesis work aimed to optimize the heterologous expression of human O-GlcNAc in E. coli. The growth condition turned out to affect protein formation significantly. The isolation was developed by combining different compounds, showing interesting behaviours of the enzyme.