Presentation Title

CANCER GENE THERAPY TARGETED TOWARDS METHIONINE METABOLISM: CHARACTERIZATION OF METHIONINE GAMMA LYASE-DEAMINASE (MGLD

Location

POSTER PRESENTATIONS

Format

Event

Start Date

12-2-2016 12:00 AM

Abstract

Objective. Our objective was to characterize the recombinant, overexpressed, purified Mgld enzyme in order to better use Mgld as a therapeutic target for cancer and related diseases. Background. Methionine is a key nutrient that is activated into sadenosylmethionine (SAM) which is the universal donor for various intracellular methylations. Venkatachalam et al., studied the effects of cytoplasmic methionine deprivation using methionine degrading enzyme methionine gammalyase-deaminase (Mgld) gene constructs that was transfected on to various cancer cell types. It was reported that upon Mgld transfection there was either severe cell aggregation and/or moderate (10-30%) cell death depending on the cancer cell types. The vector that causes the expression of Mgld protein in the cytoplasm is called C-Mgld. Venkatachalam et al., have molecularly cloned the Mgld into a new vector that has nuclear localization signal (NLS) DNA sequence upstream to Mgld gene (NLS-Mgld). Characterizing the Mgld would facilitate the optimization of the target for cancer cell therapeutics. Methods. Mgld gene was cloned into bacterial overexpression vector was transformed into BL-21 strain of E.coli. The recombinant colony was selected with kanamycin and the colonies were grown in bulk in special growth medium (Terrific Broth, TB) that contained 50 μg/mL. The culture was then induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) and the cells were grown further for 7-9 hours. The culture media was centrifuged, pellet lysed with buffer that contained protease inhibitor, purified by Ni+ affinity chromatography, TEV cleaved and further purified by DEAE column chromatography. Peak fractions were pyridoxal phosphate (PLP) exchanged. The purity was tested by SDS-PAGE. PLP antibody further confirmed the purity of the holoenzyme. Results. We feel the recombinant bacteria cultured in TB broth yielded higher amounts of Mgld protein. The purified PLP exchanged protein had characteristic 425 nm absorption peak. The purified protein had maximal activity at pH above 7.5. The enzyme exhibited temperature optimum between 37-55ºC. The lyase half reaction of Mgld exhibited a Km of ~0.6 mM for methionine and a Vmax of 58 μMols/min. The overall reaction of lyase-deaminase activity in forming α- ketobutyrate exhibited Km of 1.0 mM, Vmax of 5.27 μmol min-1mg-1, and a monomeric kcat/Km of 3729.3 M-1s-1. Conclusion. Mgld enzyme cleaves and deaminates methionine into methylthiol, ammonia and α-ketobutyrate at an appreciable rate that would dwindle intracellular methionine significantly in cancer cells while it is transfected.Therefore, we conclude persistent expression of Mgld in cancer cells would hamper cancer cell division. Hence, we feel Mgld gene is good therapeutic target for cancer cell death. Grants. President's Faculty Research and Development Grant 2015-2016

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Feb 12th, 12:00 AM

CANCER GENE THERAPY TARGETED TOWARDS METHIONINE METABOLISM: CHARACTERIZATION OF METHIONINE GAMMA LYASE-DEAMINASE (MGLD

POSTER PRESENTATIONS

Objective. Our objective was to characterize the recombinant, overexpressed, purified Mgld enzyme in order to better use Mgld as a therapeutic target for cancer and related diseases. Background. Methionine is a key nutrient that is activated into sadenosylmethionine (SAM) which is the universal donor for various intracellular methylations. Venkatachalam et al., studied the effects of cytoplasmic methionine deprivation using methionine degrading enzyme methionine gammalyase-deaminase (Mgld) gene constructs that was transfected on to various cancer cell types. It was reported that upon Mgld transfection there was either severe cell aggregation and/or moderate (10-30%) cell death depending on the cancer cell types. The vector that causes the expression of Mgld protein in the cytoplasm is called C-Mgld. Venkatachalam et al., have molecularly cloned the Mgld into a new vector that has nuclear localization signal (NLS) DNA sequence upstream to Mgld gene (NLS-Mgld). Characterizing the Mgld would facilitate the optimization of the target for cancer cell therapeutics. Methods. Mgld gene was cloned into bacterial overexpression vector was transformed into BL-21 strain of E.coli. The recombinant colony was selected with kanamycin and the colonies were grown in bulk in special growth medium (Terrific Broth, TB) that contained 50 μg/mL. The culture was then induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) and the cells were grown further for 7-9 hours. The culture media was centrifuged, pellet lysed with buffer that contained protease inhibitor, purified by Ni+ affinity chromatography, TEV cleaved and further purified by DEAE column chromatography. Peak fractions were pyridoxal phosphate (PLP) exchanged. The purity was tested by SDS-PAGE. PLP antibody further confirmed the purity of the holoenzyme. Results. We feel the recombinant bacteria cultured in TB broth yielded higher amounts of Mgld protein. The purified PLP exchanged protein had characteristic 425 nm absorption peak. The purified protein had maximal activity at pH above 7.5. The enzyme exhibited temperature optimum between 37-55ºC. The lyase half reaction of Mgld exhibited a Km of ~0.6 mM for methionine and a Vmax of 58 μMols/min. The overall reaction of lyase-deaminase activity in forming α- ketobutyrate exhibited Km of 1.0 mM, Vmax of 5.27 μmol min-1mg-1, and a monomeric kcat/Km of 3729.3 M-1s-1. Conclusion. Mgld enzyme cleaves and deaminates methionine into methylthiol, ammonia and α-ketobutyrate at an appreciable rate that would dwindle intracellular methionine significantly in cancer cells while it is transfected.Therefore, we conclude persistent expression of Mgld in cancer cells would hamper cancer cell division. Hence, we feel Mgld gene is good therapeutic target for cancer cell death. Grants. President's Faculty Research and Development Grant 2015-2016