Methionine Gamma Lyase-Deaminase (Mgld) type enzymes in previous literature have shown great potential for targeted gene therapy against cancer. Mechanistically this enzyme breaks down methionine and plays a pivotal role in sulfur amino acid catabolism in bacteria. This novel strain of Mgld enzyme, first characterized from bacterial cultures found in gingiva tissue, has proven difficult to purify and a novel strategy has been devised. Recombinant E.coli containing Mgld plasmid was grown in terrific broth for 22 hours and induced with IPTG between 5-9 hours. This culture conditions yielded very good expression of Mgld as opposed to Luria broth. Mgld recombinant protein was then purified by nickel affinity column chromatography and eluted with an imidazole gradient of 20-200 mM. The peak fractions from (100 mM imidazole) eluate were then pooled, concentrated by 0.2 mM Amicon filters and cleaved by TEV protease (1:100) dilution at 40C. The cleaved protein was then further purified by DEAE ion exchange column chromatography and eluted with PBS buffer containing 0.1-0.7 mM NaCl gradient. The Mgld that eluted at ~0.31 mM NaCl was 98% pure tested by SDS-PAGE. The purified protein was characterized first colormetrically at 425nm and by DTNB assay for lyase half reaction. Since the degradative product methyl-thiol is highly volatile (Bp ~4.6), a mineral oil overlay on the assay wells improved the quantitation of the protein significantly. The lyase half reaction of Mgld exhibited a Km of 0.7 mM for methionine and a Vmax of 58 mMols/min. Further investigation is needed to study the effects of this new purified strain of Mgld enzyme and its effect on cancer cell lines.

Cancer gene therapy: Purification and characterization of lyase activity of methionine gamma lyase-deaminase (Mgld) from Porphyromonas gingivalis

251st National Meeting (2016)

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