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Planta Med 2017; 83(10): 862-869
DOI: 10.1055/s-0043-103964
Natural Product Chemistry and Analytical Studies
Original Papers
Georg Thieme Verlag KG Stuttgart · New York

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

Jang-Ho Ha   *
1   Sustainable Resources Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea
,
Ha-Neul Kim   *
2   Department of Pharmaceutical Engineering, Dongshin University, Naju, Jeonnam, Korea
,
Ki-Beom Moon
1   Sustainable Resources Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea
,
Jae-Heung Jeon
1   Sustainable Resources Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea
,
Dai-Hyun Jung
3   Medical Nanomaterial Institute, Bio-FD&C, Hwasun, Jeonnam 58141, Korea
,
Su-Jung Kim
3   Medical Nanomaterial Institute, Bio-FD&C, Hwasun, Jeonnam 58141, Korea
,
Hugh S. Mason
4   Biodesign Institute, School of Life Sciences, Arizona State University, Tempe, AZ, USA
,
Seo-Yeon Shin
2   Department of Pharmaceutical Engineering, Dongshin University, Naju, Jeonnam, Korea
,
Hyun-Soon Kim
1   Sustainable Resources Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea
,
Kyung-Mok Park
2   Department of Pharmaceutical Engineering, Dongshin University, Naju, Jeonnam, Korea
› Institutsangaben
Weitere Informationen

Publikationsverlauf

received 25. September 2016
revised 26. Januar 2017

accepted 07. Februar 2017

Publikationsdatum:
01. März 2017 (online)

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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.


Supporting Information

A schematic representation of the expression vector used in this study is available as Supporting Information.

 

  • 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
    CrossrefPubMedGoogle Scholar
  • 2 Burgess WH, Maciag T. The heparin-binding (fibroblast) growth factor family of proteins. Annu Rev Biochem 1989; 58: 575-606
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 11 Sabalza M, Christou P, Capell T. Recombinant plant-derived pharmaceutical proteins: current technical and economic bottlenecks. Biotechnol Lett 2014; 36: 2367-2379
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 13 Chan HT, Daniell H. Plant-made oral vaccines against human infectious diseases – Are we there yet?. Plant Biotechnol J 2015; 13: 1056-1070
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 16 Fischer R, Stoger E, Schillberg S, Christou P, Twyman R. Plant-based production of biopharmaceuticals. Curr Opin Plant Biol 2004; 7: 152-158
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 22 Sainsbury F, Lomonossoff GP. Transient expressions of synthetic biology in plants. Curr Opin Plant Biol 2014; 19: 1-7
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 28 Gilchrest BA. Skin aging and photoaging: an overview. J Am Acad Dermatol 1989; 21: 610-613
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 35 Aspelund MT, Glatz CE. Purification of recombinant plant-made proteins from corn extracts by ultrafiltration. J Memb Sci 2010; 353: 103-110
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    CrossrefPubMedGoogle Scholar
  • 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
    PubMedGoogle Scholar