Recombinant Human Acidic Fibroblast Growth Factor (aFGF) Expressed in Nicotiana benthamiana Potentially Inhibits Skin Photoaging

Abstract

Responding to the need for recombinant acidic fibroblast growth factor in the pharmaceutical and cosmetic industries, we established a scalable expression system for recombinant human aFGF using transient and a DNA replicon vector expression in Nicotiana benthamiana. Recombinant human-acidic fibroblast growth factor was recovered following Agrobacterium infiltration of N. benthamiana. The optimal time point at which to harvest recombinant human acidic fibroblast growth factor expressing leaves was found to be 4 days post-infiltration, before necrosis was evident. Commassie-stained SDS-PAGE gels of His-tag column eluates, concentrated using a 10 000 molecular weight cut-off column, showed an intense band at the expected molecular weight for recombinant human acidic fibroblast growth factor. An immunoblot confirmed that this band was recombinant human acidic fibroblast growth factor. Up to 10 µg recombinant human-acidic fibroblast growth factor/g of fresh leaves were achieved by a simple affinity purification protocol using protein extract from the leaves of agroinfiltrated N. benthamiana. The purified recombinant human acidic fibroblast growth factor improved the survival rate of UVB-irradiated HaCaT and CCD-986sk cells approximately 89 and 81 %, respectively. N. benthamiana-derived recombinant human acidic fibroblast growth factor showed similar effects on skin cell proliferation and UVB protection compared to those of Escherichia coli-derived recombinant human acidic fibroblast growth factor. Additionally, N. benthamiana-derived recombinant human acidic fibroblast growth factor increased type 1 procollagen synthesis up to 30 % as well as reduced UVB-induced intracellular reactive oxygen species generation in fibroblast (CCD-986sk) cells.

UVB is a well-known factor that causes various types of skin damage and premature aging. Therefore, the present study demonstrated that N. benthamiana-derived recombinant human acidic fibroblast growth factor effectively protects skin cell from UVB, suggesting its potential use as a cosmetic or therapeutic agent against skin photoaging.

Key words

Nicotiana benthamiana - Solanaceae - recombinant human aFGF - transient expression - DNA replicon vector - skin photoaging - UVB irradiation

^* These two authors contributed equally to this work.

• References

• 1 Pellegrini L, Burke DF, von Delft F, Mulloy B, Blundell TL. Crystal structure of fibroblast growth factor receptor ectodomain bound to ligand and heparin. Nature 2000; 407: 1029-1034
• 2 Burgess WH, Maciag T. The heparin-binding (fibroblast) growth factor family of proteins. Annu Rev Biochem 1989; 58: 575-606
• 3 Yamashita T, Yoshioka M, Itoh N. Identification of a novel fibroblast growth factor, FGF-23, preferentially expressed in the ventrolateral thalamic nucleus of the brain. Biochem Biophys Res Commun 2000; 277: 494-498
• 4 Ingber DE, Folkman J. Mechanochemical switching between growth and differentiation during fibroblast growth factor-stimulated angiogenesis in vitro: role of extracellular matrix. J Cell Biol 1989; 109: 317-330
• 5 Mellin TN, Cashen DE, Ronan JJ, Murphy BS, DiSalvo J, Thomas KA. Acidic fibroblast growth factor accelerates dermal wound healing in diabetic mice. J Invest Dermatol 1995; 104: 850-855
• 6 Liu J, Ma P, Sun Y, Yang M, Yang L, Li Y, Wu Y, Zhu X, Wang X. Expression of human acidic fibroblast growth factor in Nicotiana benthamiana with a potato-virus-X-based binary vector. Biotechnol Appl Biochem 2007; 48: 143-147
• 7 Zazo M, Lozano RM, Ortega S, Varela J, Díaz-Orejas R, Ramírez JM, Giménez-Gallego G. High-level synthesis in Escherichia coli of shortened and full-length human acidic fibroblast growth factor and purification in a form stable in aqueous solutions. Gene 1992; 113: 231-238
• 8 Wu X, Kamei K, Sato H, Sato SI, Takano R, Ichida M, Mori H, Hara S. High-level expression of human acidic fibroblast growth factor and basic fibroblast growth factor in silkworm (Bombyx mori L.) using recombinant baculovirus. Protein Expr Purif 2001; 21: 192-200
• 9 Lai H, Chen Q. Bioprocessing of plant-derived virus-like particles of Norwalk virus capsid protein under current good manufacture practice regulations. Plant Cell Rep 2012; 31: 573-584
• 10 Mayani M, McLean MD, Hall JC, Filipe CDM, Ghosh R. Recovery and isolation of recombinant human monoclonal antibody from transgenic tobacco plants. Biochem Eng J 2011; 54: 103-108
• 11 Sabalza M, Christou P, Capell T. Recombinant plant-derived pharmaceutical proteins: current technical and economic bottlenecks. Biotechnol Lett 2014; 36: 2367-2379
• 12 Moon KB, Lee J, Kang S, Kim M, Mason HS, Jeon JH, Kim HS. Overexpression and self-assembly of virus-like particles in Nicotiana benthamiana by a single-vector DNA replicon system. Appl Microbiol Biotechnol 2014; 98: 8281-8290
• 13 Chan HT, Daniell H. Plant-made oral vaccines against human infectious diseases – Are we there yet?. Plant Biotechnol J 2015; 13: 1056-1070
• 14 Fan YJ, Li W, Wang JJ, Liu JY, Yang MY, Xu D, Zhu XJ, Wang XZ. Efficient production of human acidic fibroblast growth factor in pea (Pisum sativum L.) plants by agroinfection of germinated seeds. BMC Biotechnol 2011; 11: 45
• 15 Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R. Molecular farming in plants: host systems and expression technology. Trends Biotechnol 2003; 21: 570-578
• 16 Fischer R, Stoger E, Schillberg S, Christou P, Twyman R. Plant-based production of biopharmaceuticals. Curr Opin Plant Biol 2004; 7: 152-158
• 17 Magnusdottir A, Vidarsson H, Björnsson JM, Örvar BL. Barley grains for the production of endotoxin-free growth factors. Trends Biotechnol 2013; 31: 572-580
• 18 Kim HS, Jeon JH, Lee KJ, Ko K. N-glycosylation modification of plant-derived virus-like particles: an application in vaccines. Biomed Res Int 2014; 2014: 249519
• 19 Leuzinger K, Dent M, Hurtado J, Stahnke J, Lai H, Zhou X, Chen Q. Efficient agroinfiltration of plants for high-level transient expression of recombinant proteins. J Vis Exp 2013; 77: e50521
• 20 Han L, Zhang L, Liu J, Li H, Wang Y, Hasi A. Transient expression of optimized and synthesized nattokinase gene in melon (Cucumis melo L.) fruit by agroinfiltration. Plant Biotechnol 2015; 32: 175-180
• 21 Sindarovska YR, Golovach IS, Belokurova VB, Gerasymenko IM, Sheludko YV, Kuchuk NV. Screening of plant cell culture collection for efficient host species for Agrobacterium-mediated transient expression. Cytol Genet 2014; 48: 208-217
• 22 Sainsbury F, Lomonossoff GP. Transient expressions of synthetic biology in plants. Curr Opin Plant Biol 2014; 19: 1-7
• 23 Marillonnet S, Thoeringer C, Kandzia R, Klimyuk V, Gleba Y. Systemic Agrobacterium tumefaciens – mediated transfection of viral replicons for efficient transient expression in plants. Nat Biotechnol 2005; 23: 718-723
• 24 Zhou F, Wang ML, Albert HH, Moore PH, Zhu YJ. Efficient transient expression of human GM-CSF protein in Nicotiana benthamiana using potato virus X vector. Appl Microbiol Biotechnol 2006; 72: 756-762
• 25 Cañizares MC, Liu L, Perrin Y, Tsakiris E, Lomonossoff GP. A bipartite system for the constitutive and inducible expression of high levels of foreign proteins in plants. Plant Biotechnol J 2006; 4: 183-193
• 26 Huang Z, Chen Q, Hjelm B, Arntzen C, Mason H. A DNA replicon system for rapid high-level production of virus-like particles in plants. Biotechnol Bioeng 2009; 103: 706-714
• 27 Fisher GJ, Kang S, Varani J, Bata-Csorgo Z, Wan Y, Datta S, Voorhees JJ. Mechanisms of photoaging and chronological skin aging. Arch Dermatol 2002; 138: 1462-1470
• 28 Gilchrest BA. Skin aging and photoaging: an overview. J Am Acad Dermatol 1989; 21: 610-613
• 29 Anggakusuma, Yanti. Hwang JK. Effects of macelignan isolated from Myristica fragrans Houtt. on UVB-induced matrix metalloproteinase-9 and cyclooxygenase-2 in HaCaT cells. J Dermatol Sci 2010; 57: 114-122
• 30 Svobodová A, Psotová J, Walterová D. Natural phenolics in the prevention of UV-induced skin damage. A review. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2003; 147: 137-145
• 31 Ravanti L, Heino J, López-Otín C, Kähäri VM. Induction of collagenase-3 (MMP-13) expression in human skin fibroblasts by three-dimensional collagen is mediated by p38 mitogen-activated protein kinase. J Biol Chem 1999; 274: 2446-2455
• 32 Brennan M, Bhatti H, Nerusu KC, Bhagavathula N, Kang S, Fisher GJ, Varani J, Voorhees JJ. Matrix metalloproteinase-1 is the major collagenolytic enzyme responsible for collagen damage in UV-irradiated human skin. Photochem Photobiol 2003; 78: 43-48
• 33 Chen Q, He J, Phoolcharoen W, Mason HS. Geminiviral vectors based on bean yellow dwarf virus for production of vaccine antigens and monoclonal antibodies in plants. Hum Vaccin 2011; 7: 331-338
• 34 Matsuo K, Hong JS, Tabayashi N, Ito A, Masuta C, Matsumura T. Development of Cucumber mosaic virus as a vector modifiable for different host species to produce therapeutic proteins. Planta 2007; 225: 277-286
• 35 Aspelund MT, Glatz CE. Purification of recombinant plant-made proteins from corn extracts by ultrafiltration. J Memb Sci 2010; 353: 103-110
• 36 Kim DS, Qiao L, Lee KJ, Ko K. Optimization of colorectal cancer vaccine candidate protein GA733-Fc expression in a baculovirus – insect cell system. Entomol Res 2015; 45: 39-48
• 37 Park SR, Lim CY, Kim DS, Ko K. Optimization of ammonium sulfate concentration for purification of colorectal cancer vaccine candidate recombinant protein GA733-FcK isolated from plants. Front Plant Sci 2015; 6: 1040
• 38 Sankian M, Talebi F, Moghadam M, Vahedi F, Azad FJ, Varasteh AR. Molecular cloning and expression of Cucumisin (Cuc m 1), a subtilisin-like protease of Cucumis melo in Escherichia coli . Allergol Int 2011; 60: 61-67
• 39 Adil MD, Kaiser P, Satti NK, Zargar AM, Vishwakarma RA, Tasduq SA. Effect of Emblica officinalis (fruit) against UVB-induced photo-aging in human skin fibroblasts. J Ethnopharmacol 2010; 132: 109-114
• 40 Kim MS, Oh GH, Kim MJ, Hwang JK. fucosterol inhibits matrix metalloproteinase expression and promotes type 1 procollagen production in UVB-induced HaCaT cells. Photochem Photobiol 2013; 89: 911-918
• 41 Panusa A, Zuorro A, Lavecchia R, Marrosu G, Petrucci R. Recovery of natural antioxidants from spent coffee grounds. J Agric Food Chem 2013; 61: 4162-4168
• 42 Wang KH, Lin RD, Hsu FL, Huang YH, Chang HC, Huang CY, Lee MH. Cosmetic applications of selected traditional Chinese herbal medicines. J Ethnopharmacol 2006; 106: 353-359
• 43 Miller L. Quantifying western blots without expensive commercial quantification software. Available at. http://www.lukemiller.org/journal/2007/08/quantifying-western-blots-without.html Accessed October 29, 2015