Polymerase epsilon (Polε) is responsible for leading-strand synthesis during nuclear genome replication in eukaryotes and is comprised of a large catalytic subunit that contains conserved polymerase activity and an exonuclease domain with 3’–5’ proofreading activity. Mutations in the proofreading domain of Polε have been shown to cause ultramutated phenotype and strongly contribute to tumorigenesis. Kinetic investigation of the mechanistic effects of cancer-driving mutations will provide mechanisms of infidelity that lead to tumorigenesis, allowing clarifying mechanisms of fundamental DNA polymerase activity in health and disease. The critical first step in this process, however, is to determine kinetic parameters of wild type Polε to set a baseline to which we can compare mutants. In order to achieve this, a robust expression and purification scheme for WT human Polε needs to be developed, which was the focus of this thesis work. A construct containing the catalytic subunit of human Polε has been successfully heterologously expressed in an E. coli expression system and purified to obtain an active and highly pure enzyme in the amounts sufficient for future thorough kinetic investigation.