High expression and activity of an L-methionine γ-lyase gene in Escherichia coli

HU Hai-Yan1 DU Shao-Ping1 XIA Feng-Geng1 HUANG Kui-Ying1 ZHOU Shi-Ning2

(1.Guangzhou Institute of Microbiology, Guangzhou, Guangdong, China 510663)
(2.State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China 510275)

【Abstract】[Background] In order to develop new microbial resources in the ocean, we constructed a deep-sea metagenomic library by adopting the culture-independent metagenomic technology, and carried out studies on the important genes. [Objective] To identify and highly express the methionine γ-lyase gene in Escherichia coli from the DNA library of deep-sea sediments. [Methods] The gene mgl was overexpressed by pET-28a(+) system in E. coli BL21(DE3), which was induced by isopropyl β-D-1-thiogalactopyranoside, and the expression conditions were optimized to obtain the high level of recombinant methionine lyase (rMGL). The recombinant protein was purified by affinity chromatography and the enzyme activity was determined. [Results] The product rMGL had the molecular weight consistent with the predicted 46 kD, with high L-methionine lyase activity. rMGL could use L-methionine or DL-homocysteine as substrate, while had little activity for L-cysteine or L-cystine. Its relative activity for DL-homocysteine was 1.4 times of that for L-methionine. [Conclusion] mgl gene from the deep sea metagenomic library can efficiently express rMGL using pET-28 a(+)/BL21(DE3).

【Keywords】 Metagenomic library; Idiomarina; L-methionine γ-lyase; Induced expression; Purification of recombinant protein;

【DOI】

【Funds】 Special Scientific Research Project of Guangzhou Science and Technology Plan (201607010326)

Download this article

(Translated by CHEN T)

    References

    [1] Huang YL, Lai XT, He XC, et al. Characterization of a deep-sea sediment metagenomic clone that produces water-soluble melanin in Escherichia coli [J]. Marine Biotechnology, 2009, 11 (1): 124–131

    [2] Ivanova EP, Mikhailov VV. A new family, Alteromonadaceae fam. nov., including marine proteobacteria of the genera Alteromonas, Pseudoalteromonas, Idiomarina, and Colwellia [J]. Microbiology, 2001, 70 (1): 10–17

    [3] Tanaka H, Esaki N, Soda K. Properties of L-methionine γ-lyase from Pseudomonas ovalis [J]. Biochemistry, 1997, 16 (1): 100–106

    [4] Ohigashi K, Tsunetoshi A, Ichihara K. The role of pyrioxal in methylmercaptan formation, partial purification and resolution of methioninase [J]. Medical Journal of Osaka University, 1951, 2 (2): 111–117

    [5] Kreis W, Hession C. Isolation and purification of L-methionine-α-deamino-γ-mercaptomethane-lyase (L-methioninase) from Clostridium sporogenes [J]. Cancer Research, 1973, 33 (8): 1862–1865

    [6] Nakayama T, Esaki N, Sugie K, et al. Purification of bacterial L-methionine γ-lyase [J]. Analytical Biochemistry, 1984, 138 (2): 421–424

    [7] Lockwood BC, Coombs BH. Purification and characterization of methionine γ-lyase from Trichomonas vaginalis [J]. The Biochemical Journal, 1991, 279: 675–682

    [8] Faleev NG, Troitskaya MV, Paskonova EA, et al. L-Methionine-γ-lyase in Citrobacter intermedius cells: stereochemical requirements with respect to the thiol structure [J]. Enzyme and Microbial Technology, 1996, 19 (8): 590–593

    [9] Dias B, Weimer B. Purification and characterization of L-methionine γ-lyase from Brevibacterium linens BL2 [J]. Applied and Environmental Microbiology, 1998, 64 (9): 3327–3331

    [10] Yoshimura M, Nakano Y, Yamashita Y, et al. Formation of methyl mercaptan from L-methionine by Porphyromonas gingivalis [J]. Infection and Immunity, 2000, 68 (12): 6912–6916

    [11] Tokoro M, Asai T, Kobayashi S, et al. Identification and characterization of two isoenzymes of methionine γ-lyase from Entamoeba histolytica: a key enzyme of sulfur-amino acid degradation in an anaerobic parasitic protist that lacks forward and reverse trans-sulfuration pathways [J]. The Journal of Biological Chemistry, 2003, 278 (43): 42717–42727

    [12] Manukhov IV, Mamaeva DV, Rastorguev SM, et al. A gene encoding L-methionine γ-lyase is present in Enterobacteriaceae family genomes: identification and characterization of Citrobacter freundii L-methionine γ-lyase [J]. Journal of Bacteriology, 2005, 187 (11): 3889–3893

    [13] Rébeillé F, Jabrin S, Bligny R, et al. Methionine catabolism in Arabidopsis cells is initiated by a γ-cleavage process and leads to S-methylcysteine and isoleucine syntheses [J]. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103 (42): 15687–15692

    [14] Miki K, Xu MX, An ZL, et al. Survival efficacy of the combination of the methioninase gene and methioninase in a lung cancer orthotopic model [J]. Cancer Gene Therapy, 2000, 7 (2): 332–338

    [15] Kokkinakis DM. Methionine-stress: a pleiotropic approach in enhancing the efficacy of chemotherapy [J]. Cancer Letters, 2006, 233 (2): 195–207

    [16] Peron K, Jones TN, Gauthier SA, et al. Targeting of a novel fusion protein containing methioninase to the urokinase receptor to inhibit breast cancer cell migration and proliferation [J]. Cancer Chemotherapy and Pharmacology, 2003, 52 (4): 270–276

    [17] Ma BK, Wang HB. High expression of the L-methionine γ-lyase gene in E. coli [J]. Journal of Medical Postgraduates, 2008, 21 (4): 353–355, 359 (in Chinese)

    [18] Kong CH, Xia LL, Xu SL, et al. Expression, renaturation and activity of recombinant methionine γ-lyase expressed as inclusion body in E. coli [J]. Biotechnology Bulletin, 2010 (4): 189–193 (in Chinese)

This Article

ISSN:0253-2654

CN: 11-1996/Q

Vol 46, No. 12, Pages 3225-3232

December 2019

Downloads:0

Share
Article Outline

Abstract

  • 1 Materials and methods
  • 2 Results
  • 3 Discussion
  • 4 Conclusion
  • References