2019, Number 1
Next >>
Biotecnol Apl 2019; 36 (1)
Expression of rMnSODSeq regulated by the auto-inducible dps promoter in Escherichia coli
Catur RA, Olga KAR, Retnoningrum DS
Language: English
References: 21
Page: 1201-1205
PDF size: 562.40 Kb.
ABSTRACT
Recombinant protein production has been favored in Escherichia coli by using the T7 promoter, strongly regulated by IPTG, as a strategy for high transcriptional efficiency and to regulate protein overexpression. Unfortunately, IPTG is expensive and could be toxic to cells. In this setting, auto-inducible promoters emerged for recombinant protein production, as the dps promoter, which is growth phase-dependent and active in stationary phase. Previously, the plasmid pCAD-sod construct was generated, expressing the rMnSODSeq gene expression cassette under the dps promoter, and rMnSODSeq protein was expressed in E. coli. In this work, the rMnSODSeq overproduction using pCAD-sod under the regulation of dps promoter is characterized in E. coli TOP10. The influence of the incubation time and growth medium variables was analyzed. Protein yield was determined by SDS-PAGE assisted with ImageJ software, the protein was purified using a Ni2+-NTA column, and its activity was confirmed using zymography. Lastly, plasmid stability in E. coli TOP10 grown in both Luria-Bertani (LB) and Terrific Broth (TB) media were also assessed. Optimized overproduction was attained in 200 mL culture of both LB and TB at 37 °C and 150 rpm for 24 h. Overproduction in TB provided higher cell densities, while SOD percentage of total protein in LB reached the highest values (48.70 ± 1.15 %). Protein purification yielded rMnSODSeq with electrophoretic purity above 90 % and zymography analysis confirmed that the protein showed dismutation activity. The use of dps auto-inducible promoter in pCAD-sod plasmid for rMnSODSeq overproduction in E. coli system is promising for further upscaling.
REFERENCES
El-Gayar KE. Principles of recombinant protein production, extraction and purification from bacterial strains. Int J Microbiol Allied Sci Int J Microbiol Allied Sci. 2015;(2)2:18-33.
Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol. 2014;5:172.
Dvorak P, Chrast L, Nikel PI, Fedr R, Soucek K, Sedlackova M, et al. Exacerbation of substrate toxicity by IPTG in Escherichia coli BL21(DE3) carrying a synthetic metabolic pathway. Microb Cell Fact. 2015;14:201.
Baeshen MN, Al-Hejin AM, Bora RS, Ahmed MM, Ramadan HA, Saini KS, et al. Production of biopharmaceuticals in E. coli: Current scenario and future perspectives. J Microbiol Biotechnol. 2015;25(7):953-62.
Anindyajati, Artarini AA, Riani C, Retnoningrum DS. Plasmid copy number determination by Quantitative Polymerase Chain Reaction. Sci Pharm. 2016;84(1):89-101.
Retnoningrum DS, Santika IWM, Kesuma S, Ekowati SA, Riani C. Construction and characterization of a medium copy number expression vector carrying auto-inducible dps promoter to overproduce a bacterial superoxide dismutase in Escherichia coli. Mol Biotechnol. 2019;61(4):231-40.
Gopal GJ, Kumar A. Strategies for the production of recombinant protein in Escherichia coli. Protein J. 2013;32(6):419-25.
Retnoningrum DS, Arumsari S, Artarini A, Ismaya WT. Structure-activity relationship of a recombinant hybrid Manganese superoxide dismutase of Staphylococcus saprophyticus/S. equorum. Int J Biol Macromol. 2017;98:222-7.
Indrayati ANA, Asyarie S, Suciati TRI, Retnoningrum DS. Study on the properties of purified recombinant superoxide dismutase from Staphylococcus equorum, a local isolate from Indonesia. Int J Pharm Pharm Sci. 2014;6:440-5.
Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9(7):671-5.
Altuvia S, Almiron M, Huisman G, Kolter R, Storz G. The dps promoter is activated by OxyR during growth and by IHF and sigma S in stationary phase. Mol Microbiol. 1994;13(2):265-72.
Grainger DC, Goldberg MD, Lee DJ, Busby SJ. Selective repression by Fis and H-NS at the Escherichia coli dps promoter. Mol Microbiol. 2008;68(6):1366-77.
Sethia PP, Rao KK, Noronha SB. dps’: An H2O2 inducible promoter for high level protein production in Escherichia coli. 2011 International Conference on Environment and BioScience. IPCBEE. 2011;21:110-4.
Sethia PP, Rao KK, Noronha SB. A dps promoter based expression system for improved solubility of expressed proteins in Escherichia coli. Biotechnol Bioprocess Eng. 2014;19:790-7.
Santi C. Effect of plasmid pCAD_sod and its derivatives on Escherichia coli TOP10 growth profile, sod gene expression and plasmid stability [Indonesian]; 2017.
Green MR, Sambrook J. Molecular Cloning: A Laboratory Manual, 4th ed. Cold Spring Harbor Laboratory Press,U.S., New York, United States; 2012.
Li M, Liao X, Zhang D, Du G, Chen J. Yeast extract promotes cell growth and induces production of polyvinyl alcoholdegrading enzymes. Enzyme Res. 2011;2011: 179819.
Sezonov G, Joseleau-Petit D, D’Ari R. Escherichia coli physiology in Luria-Bertani broth. J Bacteriol. 2007;189(23):8746-9.
Francis DM, Page R. Strategies to optimize protein expression in E. coli. Curr Protoc Protein Sci. 2010;5(24):1-9.
European Medicines Agency. ICH Topic Q 6 B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/ Biological Products. London: EMEA;1999.
Ding D, Liu S, Wang K, Huang L, Zhao J. Expression, purification, and characterization of Cu/ZnSOD from Panax ginseng. Molecules. 2014;19(6):8112-23.