Cloning, purification and enzymatic characterization of recombinant human Superoxide dismutase 1 expressed in Escherichia coli

LIN FENG; Yan dan dan; Chen ya wen; Fletcher Emmanuella E; shi hai feng; han bang xing; zhou yang

doi:10.18388/abp.2017_2350

LIN FENG Jiangsu University Zhejiang Institute of Freshwater Fisheries
Yan dan dan
Chen ya wen
Fletcher Emmanuella E
shi hai feng
han bang xing
zhou yang

DOI: https://doi.org/10.18388/abp.2017_2350

Keywords: superoxide dismutase 1, Escherichia coli, soluble expression, metal ions, catalytic activity

Abstract

Superoxide dismutase 1 (SOD1) is a metalloenzyme that catalyzes disproportion-action superoxide into molecular oxygen and hydrogen peroxide. In this study, the human SOD1 (hSOD1) gene was cloned, expressed, and purified. The hSOD1 gene was amplified from a pool of Bxpc3 cell cDNAs by PCR and cloned into expression vector pET-28a (+). The recombinant soluble hSOD1 was expressed in E.coli BL21 (DE3) at 37°C and purified by Nickel column affinity chromatography. The soluble hSOD1 was produced with a yield of 5.9 ug/mL medium. Considering that metal ions have a certain influence on the structure and activity of protein, we researched the influences of different concentrations of Cu²⁺ and Zn²⁺ on hSOD1 activity at induction and the time of activity detection. The results implied Cu²⁺ and Zn²⁺ can’t enhance SOD1 expression, however can improve the catalytic activity at induction. Furthermore, most of bivalent cations have an improve effect on enzyme activity at the time of detection.

Author Biography

LIN FENG, Jiangsu University Zhejiang Institute of Freshwater Fisheries

Key Laboratory of Healthy Freshwater Aquaculture, Ministry of Agriculture

References

Broering TJ, Wang H, Boatright NK, Wang Y, Baptista K, Shayan G, Garrity KA, Kayatekin C, Bosco DA, Matthews CR, Ambrosino DM, Xu Z, Babcock GJ (2013) Identification of human monoclonal antibodies specific for human SOD1 recognizing distinct epitopes and forms of SOD1. PLoS One 8: e61210. doi:10.1371/journal.pone.0061210.

Becer E, Cirakoglu A (2015) Association of the Ala16Val MnSOD gene polymorphism with plasma leptin levels and oxidative stress biomarkers in obese patients. Gene 568: 35-39. doi:10.1016/ j.gene.2015.05.009.

Banci L, Barbieri L, Bertini I, Cantini F, Luchinat E (2011) In-cell NMR in E. coli to monitor maturation steps of hSOD1. PLoS One 6: e23561. doi:10.1371/journal.pone.0023561.

Chen YL, Kan WM (2015) Down-regulation of superoxide dismutase 1 by PMA is involved in cell fate determination and mediated via protein kinase D2 in myeloid leukemia cells. Biochim Biophys Acta 1853: 2662-2675. doi:10.1016/j.bbamcr.2015.07.025.

Fridovich I (1997) Superoxide anion radical (O2-.), superoxide dismutases, and related matters. J Biol Chem 272: 18515-18517.doi: 10.1074/jbc.272.30.18515.

Geraghty P, Baumlin N, Salathe MA, Foronjy RF, D'Armiento JM (2016) Glutathione Peroxidase-1 Suppresses the Unfolded Protein Response upon Cigarette Smoke Exposure. Mediators Inflamm 2016: 9461289. doi :10.1155/2016/9461289.

Girotto S, Cendron L, Bisaglia M, Tessari I, Mammi S, Zanotti G, Bubacco L (2014) DJ-1 is a copper chaperone acting on SOD1 activation. J Biol Chem 289: 10887-10899. doi: 10.1074/jbc.M113.535112.

Huo J, Shi H, Yao Q, Chen H, Wang L, Chen K (2010) Cloning and purification of recombinant silkworm dihydrolipoamide dehydrogenase expressed in Escherichia coli. Protein Expr Purif 72: 95-100. doi:10.1016/j.pep.2010.01.014.

Huang Y, Wu Z, Zhou B (2015) hSOD1 promotes tau phosphorylation and toxicity in the Drosophila model. J Alzheimers Dis 45: 235-244.doi: 10.3233/JAD-141608.

Johnson P (2002) Antioxidant enzyme expression in health and disease: effects of exercise and hypertension. Comp Biochem Physiol C Toxicol Pharmacol 133: 493-505. doi:10.1016/S1532-0456(02)00120-5.

Kilic N, Yavuz Taslipinar M, Guney Y, Tekin E, Onuk E (2014) An investigation into the serum thioredoxin, superoxide dismutase, malondialdehyde, and advanced oxidation protein products in patients with breast cancer. Ann Surg Oncol 21: 4139-4143. doi:10.1245/s10434-014-3859-3.

Kalkan G, Seckin HY, Bas Y, Benli I, Ozyurt H, Ates O, Ozdemir A, Pancar GS (2014) Analysis of manganese superoxide dismutase (MnSOD Ala-9Val) and glutathione peroxidase (GPx1 Pro 198 Leu) gene polymorphisms in psoriasis. Arch Dermatol Res 306: 253-258. doi:10.1007/s00403-013-1427-5.

Lin C, Zeng H, Lu J, Xie Z, Sun W, Luo C, Ding J, Yuan S, Geng M, Huang M (2015) Acetylation at lysine 71 inactivates superoxide dismutase 1 and sensitizes cancer cells to genotoxic agents. Oncotarget 6: 20578-20591. doi: 10.18632/oncotarget.3987.

Leitch JM, Jensen LT, Bouldin SD, Outten CE, Hart PJ, Culotta VC (2009) Activation of Cu,Zn-superoxide dismutase in the absence of oxygen and the copper chaperone CCS. J Biol Chem 284: 21863-21871.doi: 10.1074/jbc.M109.000489.

Li HT, Jiao M, Chen J, Liang Y (2010) Roles of zinc and copper in modulating the oxidative refolding of bovine copper, zinc superoxide dismutase. Acta Biochim Biophys Sin (Shanghai) 42: 183-194.doi: 10.1093/abbs/gmq005.

Ming LJ, Valentine JS (2014) Insights into SOD1-linked amyotrophic lateral sclerosis from NMR studies of Ni(2+)- and other metal-ion-substituted wild-type copper-zinc superoxide dismutases. J Biol Inorg Chem 19: 647-657. doi:10.1007/s00775-014-1126-5.

Nordlund A, Leinartaite L, Saraboji K, Aisenbrey C, Grobner G, Zetterstrom P, Danielsson J, Logan DT, Oliveberg M (2009) Functional features cause misfolding of the ALS-provoking enzyme SOD1. Proc Natl Acad Sci U S A 106: 9667-9672. doi: 10.1073/pnas.0812046106.

Rumfeldt JA, Lepock JR, Meiering EM (2009) Unfolding and folding kinetics of amyotrophic lateral sclerosis-associated mutant Cu,Zn superoxide dismutases. J Mol Biol 385: 278-298.doi: 10.1016/j.jmb.2008.10.003.

Shih LY, Liou TH, Chao JC, Kau HN, Wu YJ, Shieh MJ, Yeh CY, Han BC (2006) Leptin, superoxide dismutase, and weight loss: initial leptin predicts weight loss. Obesity (Silver Spring) 14: 2184-2192. doi: 10.1038/oby.2006.256.

Swalley SE, Fulghum JR, Chambers SP (2006) Screening factors effecting a response in soluble protein expression: formalized approach using design of experiments. Anal Biochem 351: 122-127. doi:10.1016/j.ab.2005.11.046.

Shaw BF, Valentine JS (2007) How do ALS-associated mutations in superoxide dismutase 1 promote aggregation of the protein? Trends Biochem Sci 32: 78-85.doi: 10.1016/j.tibs.2006.12.005.

Vats P, Sagar N, Singh TP, Banerjee M (2015) Association of Superoxide dismutases (SOD1 and SOD2) and Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus. Free Radic Res 49: 17-24. doi:10.3109/10715762.2014.971782.

Vasina JA, Baneyx F (1997) Expression of aggregation-prone recombinant proteins at low temperatures: a comparative study of the Escherichia coli cspA and tac promoter systems. Protein Expr Purif 9: 211-218. doi:10.1006/prep.1996.0678.

Wittung-Stafshede P (2004) Role of cofactors in folding of the blue-copper protein azurin. Inorg Chem 43: 7926-7933.doi: 10.1021/ic049398g.

Wu CY, Steffen J, Eide DJ (2009) Cytosolic superoxide dismutase (SOD1) is critical for tolerating the oxidative stress of zinc deficiency in yeast. PLoS One 4: e7061. doi: 10.1371/ journal.pone.0007061.

Yoon EJ, Park HJ, Kim GY, Cho HM, Choi JH, Park HY, Jang JY, Rhim HS, Kang SM (2009) Intracellular amyloid beta interacts with SOD1 and impairs the enzymatic activity of SOD1: implications for the pathogenesis of amyotrophic lateral sclerosis. Exp Mol Med 41:611-617.doi: 10.3858/emm. 2009.41.9.067.