Purification and characterization of recombinant mouse thymidylate synthase

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Thymidylate synthase (TS) is a critical enzyme in DNA synthesis and a target for cancer chemotherapy.
• Understanding the enzyme's structure-function relationship and its interactions with inhibitors is crucial for drug development.

Purpose of the Study:

• To purify and characterize recombinant mouse thymidylate synthase (TS).
• To investigate the enzyme's stability, optimal activity conditions, and interaction with its substrate and inhibitors.
• To elucidate the binding mechanisms of inhibitors using biophysical techniques.

Main Methods:

• High-level expression of recombinant mouse TS in Escherichia coli.
• Rapid three-step purification procedure.
• Enzyme activity assays, thermal inactivation studies, and pH profile analysis.
• UV/VIS spectroscopy and 19F-NMR spectroscopy to study enzyme-inhibitor interactions.

Main Results:

• Mouse TS was purified to homogeneity with high yield, requiring Triton X-100 and glycerol for stability.
• The enzyme exhibited optimal activity at pH 7.0 and showed pH-dependent thermal stability.
• Binding studies revealed pH-dependent dissociation constants for the inhibitory ternary complex.
• Spectroscopic analyses provided insights into the formation and characteristics of enzyme-binary and enzyme-ternary complexes.

Conclusions:

• Recombinant mouse TS can be efficiently purified and is stable under specific conditions.
• The study provides detailed characterization of mouse TS activity, stability, and inhibitor binding.
• Biophysical techniques successfully elucidated the molecular mechanisms of enzyme-inhibitor interactions, relevant for antifolate drug design.