Abstract
As the most important member of antioxidant defense system, human Cu,Zn superoxide dismutase (hCu,Zn-SOD) protects cells against the free radicals produced by aerobic metabolism. hCu,Zn-SOD has been widely used in food, cosmetic and medicine industry due to its health benefits and therapeutic potentials. However, a more extensive application of hCu,Zn-SOD is limited by the challenge of expensive and low production of high-activity hCu,Zn-SOD in large scale. In this study, the codon-optimized hCu,Zn-SOD gene was synthesized, cloned into pET-28a( +) and transformed into Escherichia coli BL21(DE3). After induction with IPTG or lactose, hCu,Zn-SOD was highly expressed as soluble form in LB medium with 800 μM Cu2+ and 20 μM Zn2+ at 25 °C. The recombinant hCu,Zn-SOD was efficiently purified by nickel affinity chromatography. Through optimization of fed-batch fermentation conditions, 342 mg purified hCu,Zn-SOD was obtained from 1 L cultures fermented in a 3-L bioreactor. Furthermore, the recombinant hCu,Zn-SOD retained the enzymatic specific activity of 46,541 U/mg. This study has opened up an effective avenue for industrial production of hCu,Zn-SOD through microbial fermentation in the future.
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References
Battistoni A, CarrìMT MAP, Rotilio G (1992) Temperature-dependent protein folding in vivo –lower growth temperature increases yield of two genetic variants of Xenopus laevis Cu, Zn superoxide dismutase in Escherichia coli. Biochem Biophys Res Commun 186(3):1339–1344. https://doi.org/10.1016/S0006-291X(05)81553-0
Carrì MT, Battistoni A, Polizio F, Desideri A, Rotilio G (1994) Impaired copper binding by the H46R mutant of human Cu, Zn superoxide dismutase, involved in amyotrophic lateral sclerosis. FEBS Lett 356:314–316. https://doi.org/10.1016/0014-5793(94)01295-4
Castan A, Enfors SO (2000) Characteristics of a DO-controlled fed-batch culture of Escherichia coli. Bioproc Eng 22(6):509–515. https://doi.org/10.1007/s004499900094
Francis DM, Page R (2010) Strategies to optimize protein expression in E coli. Curr Protoc Protein Sci 61(1):5241–52429. https://doi.org/10.1002/0471140864.ps0524s61
Fridovich I (1983) Superoxide radical: an endogenous toxicant. Annu Rev Pharmacol Toxicol 23:239–257. https://doi.org/10.1146/annurev.pa.23.040183.001323
Fujii J, Myint T, Seo HG, Kayanoki Y, Ikeda Y, Taniguchi N (1995) Characterization of wild-type and amyotrophic lateral sclerosis-related mutant Cu, Zn-superoxide dismutases overproduced in baculovirus-infected insect cells. J Neurochem 64:1456–1461. https://doi.org/10.1046/j.1471-4159.1995.64041456.x
Hallewell RA, Laria I, Tabrizi A, Carlin G, Getzoff ED, Tainer JA, Cousens LS, Mullenbach GT (1989) Genetically engineered polymers of human CuZn superoxide dismutase. J Biol Chem 264(9):5260–5268. https://doi.org/10.1016/0014-5793(89)80289-3
Hallewell RA, Masiarz FR, Najarian RC, Puma JP, Quiroga MR, Randolph A, Sanchez-Pescador R, Scandella CJ, Smith B, Steimer KS, Mullenbach GT (1985) Human Cu/Zn superoxide dismutase cDNA: isolation of clones synthesising high levels of active or inactive enzyme from an expression library. Nucleic Acids Res 13(6):2017–2034. https://doi.org/10.1093/nar/13.6.201
Hartman JR, Geller T, Yavin Z, Bartfeld D, Kanner D, Aviv H, Gorecki M (1986) High-level expression of enzymatically active human Cu/Zn superoxide dismutase in Escherichia coli. Proc Natl Acad Sci 83(19):7142–7146. https://doi.org/10.1073/pnas.83.19.7142
Hayward LJ, Rodriguez JA, Kim JW, Tiwari A, Goto JJ, Cabelli DE, Valentine JS, Brown RHJ (2002) Decreased metallation and activity in subsets of mutant superoxide dismutases associated with familial amyotrophic lateral sclerosis. J Biol Chem 277:15923–15931. https://doi.org/10.1074/jbc.M112087200
Huang P, Feng L, Oldham EA, Keating MJ, Plunkett W (2000) Superoxide dismutase as a target for the selective killing of cancer cells. Nature 407(6802):390–395. https://doi.org/10.1038/35030140
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(1):95–100. https://doi.org/10.1016/j.pep.2010.01.014
Johnson P (2002) Antioxidant enzyme expression in health and disease: effects of exercise and hypertension. Comp Biochem Physiol C Toxicol Pharmacol 133(4):493–505. https://doi.org/10.1016/S1532-0456(02)00120-5
Kamran J, Jose MA, Zia F, Saima W, Malik ZA, Syed SY (2016) Engineered production of short chain fatty acid in Escherichia coli using fatty acid synthesis pathway. PLoS ONE 11(7):e0160035. https://doi.org/10.1371/journal.pone.0160035
Kilic N, Taslipinar YM, 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(13):4139–4143. https://doi.org/10.1245/s10434-014-3859-3
Kim Y, Jeon YJ, Ryu K, Kim TY (2017) Zinc(II) ion promotes anti-inflammatory effects of rhSOD3 by increasing cellular association. BMB Rep 50(2):85–90. https://doi.org/10.5483/BMBRep.2017.50.2.150
Lee SY (1996) High cell-density culture of Escherichia coli. Trends Biotechnol 14(3):98–105. https://doi.org/10.1016/0167-7799(96)80930-9
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 42(3):183–194. https://doi.org/10.1093/abbs/gmq005
Lin F, Yan D, Chen Y, Emmanuella FE, Shi H, Han B, Zhou Y (2018) Cloning, purification and enzymatic characterization of recombinant human superoxide dismutase 1 (hSOD1) expressed in Escherichia coli. Acta Biochim Pol 65(2):235–240. https://doi.org/10.18388/abp.2017_2350
Li ZP, Zhang X, Tan TW (2006) Lactose-induced production of human soluble B lymphocyte stimulator (hsBLyS) in E coli with different culture strategies. Biotechnol Lett 28(7):477–483. https://doi.org/10.1007/s10529-006-0002-y
Liu JR, Liu JG, Zhao XY, Gu YJ (2005) Purification and renaturation of recombinant human Cu, Zn-SOD by metal-chelating affinity chromatography. Sheng Wu Gong Cheng Xue Bao 21(6):993–997. https://doi.org/10.3321/j.issn:1000-3061.2005.06.025
Liu WC, Gong T, Wang QH, Liang X, Chen JJ, Zhu P (2016) Scaling-up fermentation of pichia pastoris to demonstration-scale using new methanol-feeding strategy and increased air pressure instead of pure oxygen supplement. Sci Rep 6:18439. https://doi.org/10.1038/srep18439
Lu R, Zhang T, Wu D, He Z, Jiang L, Zhou M, Cheng Y (2018) Production of functional human Cu, Zn-SOD and EC-SOD in bitransgenic cloned goat milk. Transgenic Res 27(4):343–354. https://doi.org/10.1007/s11248-018-0080-3
Marisch K, Bayer K, Cserjan-Puschmann M, Luchner M, Striedner G (2013) Evaluation of three industrial Escherichia coli strains in fed-batch cultivations during high-level SOD protein production. Microb Cell Fact 12:1–11. https://doi.org/10.1186/1475-2859-12-58
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 106(24):9667–9672. https://doi.org/10.1073/pnas.0812046106
Park DH, Yoon SYH, Nam HG, Park JM (2002) Expression of functional human-cytosolic Cu/Zn superoxide dismutase in transgenic tobacco. Biotechnol Lett 24:681–686. https://doi.org/10.1023/a:1015273714571
Perry JJP, Shin DS, Getzoff ED, Tainer JA (2010) The structural biochemistry of the superoxide dismutases. Biochim Biophys Acta 1804:245–262. https://doi.org/10.1016/j.bbapap.2009.11.004
Riesenberg D, Guthke R (1999) High-cell-density cultivation of microorganisms. Appl Microbiol Biotechnol 51(4):422–430. https://doi.org/10.1007/s002530051412
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(1):278–298. https://doi.org/10.1016/j.jmb.2008.10.003
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(1):122–127. https://doi.org/10.1016/j.ab.2005.11.046
Takeshima Y, Takatsugu N, Sugiura M, Hagiwara H (1994) High-level expression of human superoxide dismutase in the cyanobacterium Anacystis nidulans 6301. Proc Natl Acad Sci 91(21):9685–9689. https://doi.org/10.1073/pnas.91.21.9685
Tolmasoff JM, Ono T, Cutler RG (1980) Superoxide dismutase: correlation with life-span and specific metabolic rate in primate species. Proc Natl Acad Sci 77(5):2777–2781. https://doi.org/10.1073/pnas.77.5.2777
Tripathi NK, Sathyaseelan K, Jana AM, Rao PVL (2009) High yield production of heterologous proteins with Escherichia coli. Def Sci J 59(2):137–146. https://doi.org/10.14429/dsj.59.1501
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(2):211–218. https://doi.org/10.1006/prep.1996.0678
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(1):17–24. https://doi.org/10.3109/10715762.2014.971782
Wittung-Stafshede P (2004) Role of cofactors in folding of the blue-copper protein azurin. Inorg Chem 43(25):7926–7933. https://doi.org/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. https://doi.org/10.1371/journal.pone.0007061
Wurm DJ, Veiter L, Ulonska S, Eggenreich B, Herwig C, Spadiut O (2016) The E coli pET expression system revisited-mechanistic correlation between glucose and lactose uptake. Appl Microbiol Biotechnol 100(20):8721–8729. https://doi.org/10.1007/s00253-016-7620-7
Yoo HY, Kim SS, Rho HM (1999) Overexpression and simple purification of human superoxide dismutase (SOD1) in yeast and its resistance to oxidative stress. J Biotechnol 68:29–35. https://doi.org/10.1016/S0168-1656(98)00188-6
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This work is supported by grants from the Drug Innovation Major Project (2018ZX09711001-006) and CAMS Innovation Fund for Medical Sciences (CIFMS) (2019-I2M-005).
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YJL and ZP designed this project. LXL and YJL performed the experiments. All the authors analyzed the data. YJL wrote the manuscript. All authors read and approved the final manuscript.
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Yang, JL., Li, XL., Jiang, FL. et al. High-level soluble expression of human Cu,Zn superoxide dismutase with high activity in Escherichia coli. World J Microbiol Biotechnol 36, 106 (2020). https://doi.org/10.1007/s11274-020-02883-6
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DOI: https://doi.org/10.1007/s11274-020-02883-6