Abstract
Human copper/zinc superoxide dismutase (CuZn-SOD) and extracellular superoxide dismutase (EC-SOD) are two superoxide dismutases that scavenge reactive oxygen species (ROS). Their biological role of eliminating oxidative stress caused by excessive ROS levels in living organisms has been utilized in medical treatment, preventing skin photoaging and food preservation. In this study, we employed two sequences that encode human CuZn-SOD and EC-SOD, along with goat beta-casein 5′ and 3′ regulatory elements, to construct mammary gland-specific expression vectors. Bitransgenic goats were generated using somatic cell nuclear transfer (SCNT), which employed co-transfection to generate bitransgenic goat fetal fibroblast cells as donor cells, and the expression of human CuZn-SOD and EC-SOD and their biological activities were assayed in the milk. PCR and Southern blot analysis confirmed that the cloned goat harbors both hCuZn-SOD and hEC-SOD transgenes. rhCuZn-SOD and rhEC-SOD were expressed in the mammary glands of bitransgenic goat, as determined by western blotting. The expression levels were 100.14 ± 5.09 mg/L for rhCuZn-SOD and 279.10 ± 5.38 mg/L for rhEC-SOD, as determined using ELISA. A total superoxide dismutase assay with WST-8 indicates that the biological activity of rhCuZn-SOD and rhEC-SOD in goat milk is 1451 ± 136 U/mL. The results indicate that two expression vectors can simultaneously transfect goat fetal fibroblast cells as donor cells to produce transgenic goats by SCNT, and the CuZn-SOD and EC-SOD proteins secreted in the mammary glands showed biological activity. The present study thus describes an initial step in the production of recombinant human SODs that may potentially be used for therapeutic purposes.
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References
Abdul-Rahman B, Ailor E, Jarvis D, Betenbaugh M, Lee YC (2002) Beta-(1→4)-galactosyltransferase activity in native and engineered insect cells measured with time-resolved europium fluorescence. Carbohydr Res 337:2181–2186
Altmann F, Kornfeld G, Dalik T, Staudacher E, Glossl J (1993) Processing of asparagine-linked oligosaccharides in insect cells. N-acetylglucosaminyltransferase I and II activities in cultured lepidopteran cells. Glycobiology 3:619–625
Bafana A, Dutt S, Kumar A, Kumar S, Ahuja PS (2011) The basic and applied aspects of superoxide dismutase. J Mol Catal B Enzym 68:129–138
Balbas P (2001) Understanding the art of producing protein and nonprotein molecules in Escherichia coli. Mol Biotechnol 19:251–267
Baneyx F, Mujacic M (2004) Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol 22:1399–1408. https://doi.org/10.1038/nbt1029
Berridge MJ (2016) Vitamin D, reactive oxygen species and calcium signalling in ageing and disease. Philos Trans R Soc Lond B Biol Sci. https://doi.org/10.1098/rstb.2015.0434
Bickers DR, Athar M (2006) Oxidative stress in the pathogenesis of skin disease. J Invest Dermatol 126:2565–2575. https://doi.org/10.1038/sj.jid.5700340
Byun SJ et al (2013) Human extracellular superoxide dismutase (EC-SOD) expression in transgenic chicken. BMB Rep 46:404–409
Chen H-L et al (2006) Production and characterization of human extracellular superoxide dismutase in the methylotrophic yeast Pichia pastoris. J Agric Food Chem 54:8041–8047
Cheng Y et al (2012) Hybrid expression cassettes consisting of a milk protein promoter and a cytomegalovirus enhancer significantly increase mammary-specific expression of human lactoferrin in transgenic mice. Mol Reprod Dev 79:573–585
Crapo JD, Oury T, Rabouille C, Slot JW, Chang LY (1992) Copper, zinc superoxide dismutase is primarily a cytosolic protein in human cells. Proc Natl Acad Sci USA 89:10405–10409
Cui D et al (2015) Generation of bi-transgenic pigs overexpressing human lactoferrin and lysozyme in milk. Transgenic Res 24:365–373. https://doi.org/10.1007/s11248-014-9835-7
Dobie KW et al (1996) Variegated transgene expression in mouse mammary gland is determined by the transgene integration locus. Proc Natl Acad Sci USA 93:6659–6664
Douin V, Bornes S, Creancier L, Rochaix P, Favre G, Prats AC, Couderc B (2004) Use and comparison of different internal ribosomal entry sites (IRES) in tricistronic retroviral vectors. BMC Biotechnol. https://doi.org/10.1186/1472-6750-4-16
Durocher Y, Butler M (2009) Expression systems for therapeutic glycoprotein production. Curr Opin Biotechnol 20:700–707
Emerman M, Temin HM (1986) Comparison of promoter suppression in avian and murine retrovirus vectors. Nucleic Acids Res 14:9381–9396
Fan J, Watanabe T (2003) Transgenic rabbits as therapeutic protein bioreactors and human disease models. Pharmacol Ther 99:261–282
Fliedl L, Grillari J, Grillari-Voglauer R (2015) Human cell lines for the production of recombinant proteins: on the horizon. N Biotechnol 32:673–679
Fu Z, Baker D, Cheng A, Leighton J, Appelbaum E, Aon J (2016) Characterization of a Saccharomyces cerevisiae fermentation process for production of a therapeutic recombinant protein using a multivariate bayesian approach. Biotechnol Prog 32:799–812
Gonzalez-Nicolini V, Fussenegger M (2005) A novel binary adenovirus-based dual-regulated expression system for independent transcription control of two different transgenes. J Gene Med 7:1573–1585
Gupta SK, Shukla P (2016) Advanced technologies for improved expression of recombinant proteins in bacteria: perspectives and applications. Crit Rev Biotechnol 36:1089–1098
He H-J, Yuan Q-S, Yang G-Z, Wu X-F (2002) High-level expression of human extracellular superoxide dismutase in Escherichia coli and insect cells. Protein Expr Purif 24:13–17
Houdebine L-M (2009) Production of pharmaceutical proteins by transgenic animals. Comp Immunol Microbiol Infect Dis 32:107–121
Jacobs PP, Callewaert N (2009) N-glycosylation engineering of biopharmaceutical expression systems. Curr Mol Med 9:774–800
Jankowsky JL, Slunt HH, Ratovitski T, Jenkins NA, Copeland NG, Borchelt DR (2001) Co-expression of multiple transgenes in mouse CNS: a comparison of strategies. Biomol Eng 17:157–165
Jomova K, Valko M (2011) Advances in metal-induced oxidative stress and human disease. Toxicology 283:65–87. https://doi.org/10.1016/j.tox.2011.03.001
Khan AH, Bayat H, Rajabibazl M, Sabri S, Rahimpour A (2017) Humanizing glycosylation pathways in eukaryotic expression systems. World J Microbiol Biotechnol 33:4. https://doi.org/10.1007/s11274-016-2172-7
Kim H, Yoo SJ, Kang HA (2015) Yeast synthetic biology for the production of recombinant therapeutic proteins. FEMS Yeast Res 15:1–16
Kogelberg H et al (2007) Clearance mechanism of a mannosylated antibody-enzyme fusion protein used in experimental cancer therapy. Glycobiology 17:36–45. https://doi.org/10.1093/glycob/cwl053
Kwon MJ, Kim B, Lee YS, Kim TY (2012) Role of superoxide dismutase 3 in skin inflammation. J Dermatol Sci 67:81–87. https://doi.org/10.1016/j.jdermsci.2012.06.003
Lai SL, Lee T (2006) Genetic mosaic with dual binary transcriptional systems in Drosophila. Nat Neurosci 9:703–709. https://doi.org/10.1038/nn1681
Lee EU, Roth J, Paulson JC (1989) Alteration of terminal glycosylation sequences on N-linked oligosaccharides of Chinese hamster ovary cells by expression of beta-galactoside alpha 2,6-sialyltransferase. J Biol Chem 264:13848–13855
Lee YS, Choi JH, Lee JH, Lee HW, Lee W, Kim WT, Kim TY (2016) Extracellular superoxide dismutase ameliorates house dust mite-induced atopic dermatitis-like skin inflammation and inhibits mast cell activation in mice. Exp Dermatol 25:630–635. https://doi.org/10.1111/exd.13028
Leitner LM, Wilson RJ, Yan Z, Godecke A (2017) Reactive oxygen species/nitric oxide mediated inter-organ communication in skeletal muscle wasting diseases. Antioxid Redox Signal 26:700–717
Li H, Liu Q, Cui K, Liu J, Ren Y, Shi D (2013) Expression of biologically active human interferon alpha 2b in the milk of transgenic mice. Transgenic Res 22:169–178
Lima ISD, Garcez BS, Alves AA, Aquino FC, Borges LS, Carvalho WF (2016) Fat protected and profile of fatty acids goat milk: a review. Braz J Hyg Anim Sani 10:830–840. https://doi.org/10.5935/1981-2965.20160068
Lipinski D et al (2012) Expression of human growth hormone in the milk of transgenic rabbits with transgene mapped to the telomere region of chromosome 7q. J Appl Genet 53:435–442
Marklund SL (2002) Extracellular superoxide dismutase. Methods Enzymol 349:74–80
Miao L, St Clair DK (2009) Regulation of superoxide dismutase genes: implications in disease. Free Radic Biol Med 47:344–356
Milesi MA, Lacan D, Brosse H, Desor D, Notin C (2009) Effect of an oral supplementation with a proprietary melon juice concentrate (Extramel) on stress and fatigue in healthy people: a pilot, double-blind, placebo-controlled clinical trial. Nutr J 8:40. https://doi.org/10.1186/1475-2891-8-40
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
Park KY, Kim EY, Lee W, Kim T-Y, Kim WT (2016) Expression, subcellular localization, and enzyme activity of a recombinant human extra-cellular superoxide dismutase in tobacco (Nicotiana benthamiana L.). Protein Expr Purif 119:69–74
Pastuszka R, Barlowska J, Litwinczuk Z (2015) Nutritional value and health-promoting properties of goat milk. Med Weter 71:480–485
Patlevic P, Vaskova J, Svorc P Jr, Vasko L, Svorc P (2016) Reactive oxygen species and antioxidant defense in human gastrointestinal diseases. Integr Med Res 5:250–258
Reyes Escogido ML, De Leon Rodriguez A, Barba de la Rosa AP (2007) A novel binary expression vector for production of human IL-10 in Escherichia coli and bifidobacterium longum. Biotech Lett 29:1249–1253. https://doi.org/10.1007/s10529-007-9376-8
Robertson G, Garrick D, Wu W, Kearns M, Martin D, Whitelaw E (1995) Position-dependent variegation of globin transgene expression in mice. Proc Natl Acad Sci USA 92:5371–5375
Sabalza M, Christou P, Capell T (2014) Recombinant plant-derived pharmaceutical proteins: current technical and economic bottlenecks. Biotechnol Lett 36:2367–2379
Schein O, Westreich M, Shalom A (2013) Effect of intradermal human recombinant copper-zinc superoxide dismutase on random pattern flaps in rat. Head Neck 35:1265–1268. https://doi.org/10.1002/hed.23114
Shibata N et al (1996) Structure and antigenicity of the mannans of candida famata and Candida saitoana: comparative study with the mannan of Candida guilliermondii. Arch Biochem Biophys 336:49–58. https://doi.org/10.1006/abbi.1996.0531
Shrestha P, Yun J-H, Kim WT, Kim T-Y, Lee W (2016) Cloning, purification, and characterization of recombinant human extracellular superoxide dismutase in SF9 insect cells. Mol Cells 39:242–249
Simpson RB, Spielmann A, Margossian L, McKnight TD (1986) A disarmed binary vector from Agrobacterium tumefaciens functions in agrobacterium rhizogenes: frequent co-transformation of two distinct T-DNAs. Plant Mol Biol 6:403–415. https://doi.org/10.1007/BF00027133
Son Y-J, Bae J-Y, Chong S-H, Lee HS, Mo SH, Kim TY, Choe H (2010) Expression, high cell density culture and purification of recombinant EC-SOD in Escherichia coli. Appl Biochem Biotechnol 162:1585–1598
Song S et al (2016) High-level expression of a novel recombinant human plasminogen activator (rhPA) in the milk of transgenic rabbits and its thrombolytic bioactivity in vitro. Mol Biol Rep 43:775–783
Stromqvist M et al (1997) Recombinant human extracellular superoxide dismutase produced in milk of transgenic rabbits. Transgenic Res 6:271–278
Sukhapinda K, Spivey R, Shahin EA (1987) Ri-plasmid as a helper for introducing vector DNA into alfalfa plants. Plant Mol Biol 8:209–216
Swartz JR (2001) Advances in Escherichia coli production of therapeutic proteins. Curr Opin Biotechnol 12:195–201
Tatematsu K-I, Kobayashi I, Uchino K, Sezutsu H, Iizuka T, Yonemura N, Tamura T (2010) Construction of a binary transgenic gene expression system for recombinant protein production in the middle silk gland of the silkworm Bombyx mori. Transgenic Res 19:473–487
Tibell L, Hjalmarsson K, Edlund T, Skogman G, Engstrom A, Marklund SL (1987) Expression of human extracellular superoxide dismutase in Chinese hamster ovary cells and characterization of the product. Proc Natl Acad Sci USA 84:6634–6638
Tostes RC, Carneiro FS, Carvalho MHC, Reckelhoff JF (2016) Reactive oxygen species: players in the cardiovascular effects of testosterone. Am J Physiol Regul Integr Comp Physiol 310:R1–14
Viktorinova I, Wimmer EA (2007) Comparative analysis of binary expression systems for directed gene expression in transgenic insects. Insect Biochem Mol Biol 37:246–254. https://doi.org/10.1016/j.ibmb.2006.11.010
Visser RG, Hesseling-Meinders A, Jacobsen E, Nijdam H, Witholt B, Feenstra WJ (1989) Expression and inheritance of inserted markers in binary vector carrying agrobacterium rhizogenes-transformed potato (Solanum tuberosum L.). Theor Appl Genet 78:705–714
Wang Y, Zhao S, Bai L, Fan J, Liu E (2013) Expression systems and species used for transgenic animal bioreactors. Biomed Res Int 2013:580463
Wang X et al (2017) The functions of serpin-3, a negative-regulator involved in prophenoloxidase activation and antimicrobial peptides expression of Chinese oak silkworm, Antheraea pernyi. Dev Comp Immunol 69:1–11
Wilson IB, Harthill JE, Mullin NP, Ashford DA, Altmann F (1998) Core alpha1,3-fucose is a key part of the epitope recognized by antibodies reacting against plant N-linked oligosaccharides and is present in a wide variety of plant extracts. Glycobiology 8:651–661
Xingfen G, Ping Y, Yanxia Q, Jun L, Guolin Z (1998) Studies on the preparation and the stability of SOD modified by lauric acid. Wuhan Univ J Nat Sci 3:247–250. https://doi.org/10.1007/bf02827563
Xue MF, Zhao QD, Zhang W (2017) Clinical application of anti-radiation spay combined with flamigel in prevention of postoperative radioactive skin injury of breast cancer. Nurs Pract Res 14(02):97–98
Yamano N, Takahashi M, Ali Haghparast SM, Onitsuka M, Kumamoto T, Frank J, Omasa T (2016) Increased recombinant protein production owing to expanded opportunities for vector integration in high chromosome number Chinese hamster ovary cells. J Biosci Bioeng 122:226–231
Yang X, Carter MG (2007) Transgenic animal bioreactors: a new line of defense against chemical weapons? Proc Natl Acad Sci USA 104:13859–13860
Yao W, Zhao H, Shi R, Li X, Li Y, Ke C, Liu J (2017) Recombinant protein transduction domain-Cu/Zn superoxide dismutase alleviates bone cancer pain via peroxiredoxin 4 modulation and antioxidation. Biochem Biophys Res Commun 486:1143–1148. https://doi.org/10.1016/j.bbrc.2017.04.017
Yuan Y-G et al (2014) Expression of recombinant human alpha-lactalbumin in the milk of transgenic goats using a hybrid pomoter/enhancer. J Anal Methods Chem 2014:281031
Zelko IN, Mariani TJ, Folz RJ (2002) Superoxide dismutase multigene family: a comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression. Free Radic Biol Med 33:337–349
Acknowledgments
The Intergovernmental Science and Technology Cooperation Project (S2016G6252) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) supported this study. We thank Professor Yong-Cheng for providing technical assistance. We also thank the personnel of Jiangsu Provincial Research Center for Animal Transgenesis and Biopharming for assisting in this research.
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Lu, R., Zhang, T., Wu, D. et al. Production of functional human CuZn-SOD and EC-SOD in bitransgenic cloned goat milk. Transgenic Res 27, 343–354 (2018). https://doi.org/10.1007/s11248-018-0080-3
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DOI: https://doi.org/10.1007/s11248-018-0080-3