Thymidylate synthetase (TYMS) is an enzyme that catalyzes the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP). The enzyme is essential for regulating the balanced supply of the 4 DNA precursors in normal DNA replication: defects in the enzyme activity affecting the regulation process cause various biological and genetic abnormalities. Thymidylate synthase is an enzyme of about 30 to 35kDa in most species except in protozoan and plants where it exists as a bifunctional enzyme that includes a dihydrofolate reductase domain. Besides, Aspartate Aminotransferase (AST) has been identified as an interactor of TYMS, thus a binding ELISA assay was conducted to detect the interaction of recombinant human TYMS and recombinant human AST. Briefly, TYMS were diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100uL were then transferred to AST-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-TYMS pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 3 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50μL stop solution to the wells and read at 450nm immediately. The binding activity of TYMS and AST was shown in Figure 1, and this effect was in a dose dependent manner.
Figure. The binding activity of TYMS with AST.