Gene Cloning And Expression Of Methionine γ-Lyase

L-Methionineγ-lyase (MGL) can catalyse theα,γ--elimination of L-Methionine and Homocysteine and was developed as an anticancer drug as well as a diagnostic reagent for homocysteinemia. Gene cloning, expression and characterization of methionineγ-lyase from Trichomonas vaginalis were explored in this paper.Gene of L-Methionineγ-lyase was prepared by reverse PCR with RNA extracted from Trichomonas vaginalis. Expression vector pET-15b-mgl1 was constructed by the ligation of plasmid pET-15b with the gene of MGL and sequenced.Recombinant MGL was mainly expressed in the form of inclusion body in E.coli induced by IPTG The best expression condition with culrure pH 5.0, inducing temperature 27℃, inducing OD₆₀₀ 0.6, IPTG concentration 0.6mM and inducing time 5h were decided by screeneing.Mice were immunized with recombinant MGL purified by Ni²⁺ coloumn. The reaction between antiserum and wild MGL purified from Trichomonas vaginalis was tested by Western blot. The biological activity of the recombinant protein was proved. We prepared 6.58mg soluble MGL from 1L shake-flask culture with higher activity, with a 41.1% overall yield in the purification process.The Km of the recombinant MGL for L- Methionine, D L- Homocysteine, L-Cystein as substrate were 0.318mM, 2.14 mM, 1.62 mM respectively and the specific activity were 20.17,318.69,56.96 umol/min/mg protein respectively. The optimal pH for recombinant MGL was pH5.0-pH6.0 and recombinant MGL was stable up to 50℃. The results indicate that the recombinant MGL has higher activity and good thermal stability suggesting potential efficacy in future clinical trials.