Herpes simplex virus (HSV) is a double-stranded DNA virus and an important human pathogen that causes various illnesses, from clinically almost insignificant to life-threatening encephalitis. It has been shown that viral protein ICP34.5 is required for neurovirulence in mice and neurological diseases in humans. ICP34.5 is involved in the inhibition of host defense, inhibition of autophagy and disruption of IRF3 activation. Although intensively studied, the function of ICP34.5 is still not completely understood. The main aim of this study is to clone and express ICP34.5 protein from bacteria and to affinity purificate the protein for purpose of generating highly specific antibodies. For this purpose, using molecular biology techniques, we cloned ICP34.5 gene carrying the FLAG marker from the eukaryotic expression plasmid to the bacterial expression system pET32 carrying the six-histidine bilayer of the amino acids necessary for affinity purification. Success of cloning and validation of the correct DNA sequence was confirmed by sequencing. Protein expression was performed in DE3 bacteria and the resulting protein product of the expected molecular weight was obtained, however the protein level was relatively weak. Consequently, affinity purification resulted in a small amount of protein. To increase the efficiency of protein production, the ICP34.5 gene sequence was adapted to the bacterial system (codon optimiozation), which significantly affected the level of protein produced. In addition, we investigated the intracellular localization of the ICP34.5 protein expressed from the eukaryotic plasmid vector. Surprisingly, though the protein is found in the cytoplasm - cell division that describes most of the ICP34.5 protein function, most of the proteins have been found localized in nuclei and nucleolus-like structures. Our work generated the necessary vectors and optimized the heterologous ICP34.5 protein production system as part of a broader antibody-generating project for this protein, and ultimately result in a better understanding of ICP34.5 biology.