Thymidylate synthase is an intracellular enzyme critical for de novo synthesis of DNA. This function maintains the dTMP(thymidine-5-prime monophosphate) pool critical for DNA replication and repair. In cancer, expression of this protein is often elevated and becomes further elevated as a result of treatment with the most commonly used chemotherapeutic, 5-fluorouracil (5-FU). Resistance or lack of response to 5-FU is attributed to the elevation of thymidylate synthase activity. Recombinant human Thymidylate synthase protein, fused to His-tag at N-terminus, was expressed in E.coli and purified by using conventional chromatography techniques.