Abstract
Human connective tissue growth factor (CTGF) is a secreted cysteine-rich peptide that stimulates cell proliferation, migration, and extracellular matrix production during tissue development, differentiation, angiogenesis, implantation, wound healing, and fibrosis processes, with broad application in the medical and cosmetic medical fields. However, the production of CTGF is currently limited by its low yield and purity in current bioreactors. In this study, two genetically modified silkworm strains were generated harboring artificially designed CTGF-8ht and pepCTGF-8ht genes, respectively, that contain an enhanced His-tag with eight histidine residues with or without a transdermal peptide (pep). Both recombinant CTGF-8ht and pepCTGF-8ht proteins were successfully expressed in the silkworm silk gland and cocoon, and could be easily extracted and purified from the cocoon by single-affinity immunoprecipitation column chromatography, achieving a purity of more than 95%. Moreover, compared with CTGF-8ht protein, pepCTGF-8ht protein exhibited better cell proliferation activity by activating the extracellular signal-regulated kinase (ERK) pathway and enhanced hyaluronic acid synthesis activity by upregulating hyaluronan synthase 3 expression; moreover, the addition of pep significantly improved the transmembrane ability of CTGF-8ht protein. These results should help to promote the application prospects of CTGF and further guide the design and development of protein drugs from silkworm and other bioreactor systems.
Key points
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A silkworm bioreactor was optimized to produce connective tissue growth factor (CTGF)
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The transgene contained an enhanced 8-His-tag and transmembrane peptide (pep)
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Recombinant CTGF was easily purified with maintained or higher biological activity
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References
Babic AM, Chen CC, Lau LF (1999) Fisp12/mouse connective tissue growth factor mediates endothelial cell adhesion and migration through integrin alphavbeta3, promotes endothelial cell survival, and induces angiogenesis in vivo. Mol Cell Biol 19(4):2958–2966. https://doi.org/10.1128/mcb.19.4.2958
Bagis H, Aktoprakligil D, Gunes C, Arat S, Akkoc T, Cetinkaya G, Kankavi O, Taskin AC, Arslan K, Dundar M, Tsoncheva VL, Ivanov IG (2011) Expression of biologically active human interferon gamma in the milk of transgenic mice under the control of the murine whey acidic protein gene promoter. Biochem Genet 49(3-4):251–257. https://doi.org/10.1007/s10528-010-9403-7
Ball DK, Moussad EE, Rageh MA, Kemper SA, Brigstock DR (2003) Establishment of a recombinant expression system for connective tissue growth factor (CTGF) that models CTGF processing in utero. Reproduction 125(2):271–284. https://doi.org/10.1530/rep.0.1250271
Boilly B, Vercoutter-Edouart AS, Hondermarck H, Nurcombe V, Le Bourhis X (2000) FGF signals for cell proliferation and migration through different pathways. Cytokine Growth Factor Rev 11(4):295–302. https://doi.org/10.1016/s1359-6101(00)00014-9
Buckholz RG, Gleeson MA (1991) Yeast systems for the commercial production of heterologous proteins. Biotechnology (N Y) 9(11):1067–1072. https://doi.org/10.1038/nbt1191-1067
Chen W, Wang F, Tian C, Wang Y, Xu S, Wang R, Hou K, Zhao P, Yu L, Lu Z, Xia Q (2018) Transgenic silkworm-based silk gland bioreactor for large scale production of bioactive human platelet-derived growth factor (PDGF-BB) in silk cocoons. Int J Mol Sci 19(9):2533. https://doi.org/10.3390/ijms19092533
Choi JH, Lee SY (2004) Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl Microbiol Biotechnol 64(5):625–635. https://doi.org/10.1007/s00253-004-1559-9
Demain AL, Vaishnav P (2009) Production of recombinant proteins by microbes and higher organisms. Biotechnol Adv 27(3):297–306. https://doi.org/10.1016/j.biotechadv.2009.01.008
Eum WS, Kim DW, Hwang IK, Yoo KY, Kang TC, Jang SH, Choi HS, Choi SH, Kim YH, Kim SY, Kwon HY, Kang JH, Kwon OS, Cho SW, Lee KS, Park J, Won MH, Choi SY (2004) In vivo protein transduction: biologically active intact pep-1-superoxide dismutase fusion protein efficiently protects against ischemic insult. Free Radic Biol Med 37(10):1656–1669. https://doi.org/10.1016/j.freeradbiomed.2004.07.028
Fawell S, Seery J, Daikh Y, Moore C, Chen LL, Pepinsky B, Barsoum J (1994) Tat-mediated delivery of heterologous proteins into cells. Proc Natl Acad Sci U S A 91(2):664–668. https://doi.org/10.1073/pnas.91.2.664
Frazier K, Williams S, Kothapalli D, Klapper H, Grotendorst GR (1996) Stimulation of fibroblast cell growth, matrix production, and granulation tissue formation by connective tissue growth factor. J Invest Dermatol 107(3):404–411. https://doi.org/10.1111/1523-1747.ep12363389
Gleba Y, Klimyuk V, Marillonnet S (2007) Viral vectors for the expression of proteins in plants. Curr Opin Biotechnol 18(2):134–141. https://doi.org/10.1016/j.copbio.2007.03.002
Gygi D, Zumstein P, Grossenbacher D, Altwegg L, Luscher TF, Gehring H (2003) Human connective tissue growth factor expressed in Escherichia coli is a non-mitogenic inhibitor of apoptosis. Biochem Biophys Res Commun 311(3):685–690. https://doi.org/10.1016/j.bbrc.2003.10.061
Hao C, Xie Y, Peng M, Ma L, Zhou Y, Zhang Y, Kang W, Wang J, Bai X, Wang P, Jia Z (2014) Inhibition of connective tissue growth factor suppresses hepatic stellate cell activation in vitro and prevents liver fibrosis in vivo. Clin Exp Med 14(2):141–150. https://doi.org/10.1007/s10238-013-0229-6
Igarashi A, Okochi H, Bradham DM, Grotendorst GR (1993) Regulation of connective tissue growth factor gene expression in human skin fibroblasts and during wound repair. Mol Biol Cell 4(6):637–645. https://doi.org/10.1091/mbc.4.6.637
Kim MS, Song HJ, Lee SH, Lee CK (2014) Comparative study of various growth factors and cytokines on type I collagen and hyaluronan production in human dermal fibroblasts. J Cosmet Dermatol 13(1):44–51. https://doi.org/10.1111/jocd.12073
Kojima K, Kuwana Y, Sezutsu H, Kobayashi I, Uchino K, Tamura T, Tamada Y (2007) A new method for the modification of fibroin heavy chain protein in the transgenic silkworm. Biosci Biotechnol Biochem 71(12):2943–2951. https://doi.org/10.1271/bbb.70353
Lau L (2016) Cell surface receptors for CCN proteins. J Cell Commun Signal 10(2):121–127. https://doi.org/10.1007/s12079-016-0324-z
Luo X, Ma D, Wang Y, Li W, Wang C, He Y, Gu X, Li X, Zhou H, Zhang T (2016) Fusion with pep-1, a cell-penetrating peptide, enhances the transmembrane ability of human epidermal growth factor. Biosci Biotechnol Biochem 80(3):584–590. https://doi.org/10.1080/09168451
Ma S, Shi R, Wang X, Liu Y, Chang J, Gao J, Lu W, Zhang J, Zhao P, Xia Q (2014) Genome editing of BmFib-H gene provides an empty Bombyx mori silk gland for a highly efficient bioreactor. Sci Rep 4:6867. https://doi.org/10.1038/srep06867
Morris MC, Depollier J, Mery J, Heitz F, Divita G (2001) A peptide carrier for the delivery of biologically active proteins into mammalian cells. Nat Biotechnol 19(12):1173–1176. https://doi.org/10.1038/nbt1201-1173
Ogawa S, Tomita M, Shimizu K, Yoshizato K (2007) Generation of a transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon: production of recombinant human serum albumin. J Biotechnol 128(3):531–544. https://doi.org/10.1016/j.jbiotec.2006.10.019
Perbal B (2004) CCN proteins: multifunctional signalling regulators. Lancet 363(9402):62–64. https://doi.org/10.1016/S0140-6736(03)15172-0
Prochiantz A (2000) Messenger proteins: homeoproteins, TAT and others. Curr Opin Cell Biol 12(4):400–406. https://doi.org/10.1016/s0955-0674(00)00108-3
Quan T, Shao Y, He T, Voorhees JJ, Fisher GJ (2010) Reduced expression of connective tissue growth factor (CTGF/CCN2) mediates collagen loss in chronologically aged human skin. J Invest Dermatol 130(2):415–424. https://doi.org/10.1038/jid.2009.224
Royer C, Jalabert A, Da Rocha M, Grenier AM, Mauchamp B, Couble P, Chavancy G (2005) Biosynthesis and cocoon-export of a recombinant globular protein in transgenic silkworms. Transgenic Res 14(4):463–472. https://doi.org/10.1007/s11248-005-4351-4
Schwarze SR, Hruska KA, Dowdy SF (2000) Protein transduction: unrestricted delivery into all cells? Trends Cell Biol 10(7):290–295. https://doi.org/10.1016/s0962-8924(00)01771-2
Secker GA, Shortt AJ, Sampson E, Schwarz QP, Schultz GS, Daniels JT (2008) TGFbeta stimulated re-epithelialisation is regulated by CTGF and Ras/MEK/ERK signalling. Exp Cell Res 314(1):131–142. https://doi.org/10.1016/j.yexcr.2007.09.001
Shepelev MV, Kalinichenko SV, Deykin AV, Korobko IV (2018) Production of recombinant proteins in the milk of transgenic animals: current state and prospects. Acta Naturae 10(3):40–47
Shimo T, Nakanishi T, Nishida T, Asano M, Kanyama M, Kuboki T, Tamatani T, Tezuka K, Takemura M, Matsumura T, Takigawa M (1999) Connective tissue growth factor induces the proliferation, migration, and tube formation of vascular endothelial cells in vitro, and angiogenesis in vivo. J Biochem 126(1):137–145. https://doi.org/10.1093/oxfordjournals.jbchem.a022414
Shi-Wen X, Leask A, Abraham D (2008) Regulation and function of connective tissue growth factor/CCN2 in tissue repair, scarring and fibrosis. Cytokine Growth Factor Rev 19(2):133–144. https://doi.org/10.1016/j.cytogfr.2008.01.002
Tatematsu K, 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(3):473–487. https://doi.org/10.1007/s11248-009-9328-2
Toda N, Mukoyama M, Yanagita M, Yokoi H (2018) CTGF in kidney fibrosis and glomerulonephritis. Inflamm Regen 38:14. https://doi.org/10.1186/s41232-018-0070-0
Tomita M, Munetsuna H, Sato T, Adachi T, Hino R, Hayashi M, Shimizu K, Nakamura N, Tamura T, Yoshizato K (2003) Transgenic silkworms produce recombinant human type III procollagen in cocoons. Nat Biotechnol 21(1):52–56. https://doi.org/10.1038/nbt771
Tomita M, Hino R, Ogawa S, Iizuka M, Adachi T, Shimizu K, Sotoshiro H, Yoshizato K (2007) A germline transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon. Transgenic Res 16(4):449–465. https://doi.org/10.1007/s11248-007-9087-x
Vives E, Brodin P, Lebleu B (1997) A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J Biol Chem 272(25):16010–16017. https://doi.org/10.1074/jbc.272.25.16010
Wang F, Xu H, Yuan L, Ma S, Wang Y, Duan X, Duan J, Xiang Z, Xia Q (2013) An optimized sericin-1 expression system for mass-producing recombinant proteins in the middle silk glands of transgenic silkworms. Transgenic Res 22(5):925–938. https://doi.org/10.1007/s11248-013-9695-6
Wang F, Xu H, Wang Y, Wang R, Yuan L, Ding H, Song C, Ma S, Peng Z, Peng Z, Zhao P, Xia Q (2014) Advanced silk material spun by a transgenic silkworm promotes cell proliferation for biomedical application. Acta Biomater 10(12):4947–4955. https://doi.org/10.1016/j.actbio.2014.06.031
Wang F, Wang R, Wang Y, Zhao P, Xia Q (2015a) Large-scale production of bioactive recombinant human acidic fibroblast growth factor in transgenic silkworm cocoons. Sci Rep 5:16323. https://doi.org/10.1038/srep16323
Wang Y, Wang F, Wang R, Zhao P, Xia Q (2015b) 2A self-cleaving peptide-based multi-gene expression system in the silkworm Bombyx mori. Sci Rep 5:16273. https://doi.org/10.1038/srep16273
Wang F, Wang R, Wang Y, Xu H, Yuan L, Ding H, Ma S, Zhou Y, Zhao P, Xia Q (2015c) Remobilizing deleted piggyBac vector post-integration for transgene stability in silkworm. Mol Genet Genomics 290(3):1181–1189. https://doi.org/10.1007/s00438-014-0982-6
Wang Y, Wang F, Xu S, Wang R, Chen W, Hou K, Tian C, Wang F, Yu L, Lu Z, Zhao P, Xia Q (2019a) Genetically engineered bi-functional silk material with improved cell proliferation and anti-inflammatory activity for medical application. Acta Biomater 86:148–157. https://doi.org/10.1016/j.actbio.2018.12.036
Wang Y, Wang F, Xu S, Wang R, Chen W, Hou K, Tian C, Wang F, Zhao P, Xia Q (2019b) Optimization of a 2A self-cleaving peptide-based multigene expression system for efficient expression of upstream and downstream genes in silkworm. Mol Genet Genomics 294(4):849–859. https://doi.org/10.1007/s00438-019-01534-2
Wang Y, Xu S, Wang R, Chen W, Hou K, Tian C, Ji Y, Yang Q, Yu L, Lu Z, Zhao P, Xia Q, Wang F (2019c) Genetic fabrication of functional silk mats with improved cell proliferation activity for medical applications. Biomater Sci 7(11):4536–4546. https://doi.org/10.1039/c9bm01285k
de Winter P, Leoni P, Abraham D (2008) Connective tissue growth factor: structure-function relationships of a mosaic, multifunctional protein. Growth factors 26(2):80–91. https://doi.org/10.1080/08977190802025602
Xu S, Wang F, Wang Y, Wang R, Hou K, Tian C, Ji Y, Yang Q, Zhao P, Xia Q (2019) A silkworm based silk gland bioreactor for high-efficiency production of recombinant human lactoferrin with antibacterial and anti-inflammatory activities. J Biol Eng 13:61. https://doi.org/10.1186/s13036-019-0186-z
Yin Q, Liu H (2019) Connective tissue growth factor and renal fibrosis. Adv Exp Med Biol 1165:365–380. https://doi.org/10.1007/978-981-13-8871-2_17
Zhao A, Zhao T, Zhang Y, Xia Q, Lu C, Zhou Z, Xiang Z, Nakagaki M (2010) New and highly efficient expression systems for expressing selectively foreign protein in the silk glands of transgenic silkworm. Transgenic Res 19(1):29–44. https://doi.org/10.1007/s11248-009-9295-7
Funding
This work was supported by the National Natural Science Foundation of China (31501896, 32000941), Chongqing Science and Technology Commission (cstc2018jcyjAX0298), and Chongqing Natural Science Foundation (cstc2019jcyj-bshX0102).
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Q Xia and P Zhao supervised the study. Y Wang, F Wang, S Xu, R Wang, C Tian, Y Ji and Q Yang designed and performed the experiments. Y Wang wrote the manuscript. Q Xia and F Wang revised the manuscript. All authors read and approved the final manuscript.
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Wang, Y., Wang, F., Xu, S. et al. Transdermal peptide conjugated to human connective tissue growth factor with enhanced cell proliferation and hyaluronic acid synthesis activities produced by a silkworm silk gland bioreactor. Appl Microbiol Biotechnol 104, 9979–9990 (2020). https://doi.org/10.1007/s00253-020-10836-0
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DOI: https://doi.org/10.1007/s00253-020-10836-0