Chemistry and Biochemistry Department
BS in Biochemistry
Serine Hydroxymethyltransferase (SHMT) is a member of the thymidylate synthase cycle that provides pyrimidine nucleotides to actively dividing cells, including cancer cells. The other two enzymes involved in this cycle have been targets for clinical anticancer drugs, suggesting that inhibitors of SHMT may also be used as clinical anticancer drugs. In order to test potential inhibitors against SHMT, human SHMT has been overexpressed in E. coli and partially purified by selective ammonium sulfate fractionation followed by anion-exchange fast protein liquid chromatography. A coupled assay using L-allo-threonine and alcohol dehydrogenase was used to identify active fractions, which were analyzed for purity by SDS-PAGE. The affinity purification of human SHMT by Ni+2-NTA affinity chromatography trials concluded that it was not expressed as a fusion protein containing a polyhistidine affinity tag. The gene encoding for human SHMT was then cloned into a pET151/D-TOPO (Invitrogen) plasmid for better purification. A fraction of transformed colonies were screened for the cloned gene by PCR amplification and restriction enzyme digestion of isolated plasmid DNA. None of the colonies screened were found to contain the full-length hSHMT gene.