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Transgenic Research

, Volume 28, Issue 5–6, pp 601–609 | Cite as

Expression and characterization of recombinant human VEGF165 in the middle silk gland of transgenic silkworms

  • Tianyang Zhang
  • Rongpeng Liu
  • Qin Luo
  • Dawei Qu
  • Tao Chen
  • Ou Yao
  • Hanfu XuEmail author
Brief Communication
  • 118 Downloads

Abstract

Recombinant human vascular endothelial growth factor (rhVEGF) has important applications in therapeutic angiogenesis and inhibition of VEGF-mediated pathological angiogenesis. Previous studies have shown that rhVEGF can be produced in several expression systems, including Escherichia coli, yeasts, insect cells and mammalian cells. However, little is known regarding the effective production of this protein in organs of live organisms. Here, we report for the first time the expression and characterization of rhVEGF165 in the middle silk gland (MSG) of the transgenic silkworm line S1-V165. Our results confirmed that (1) rhVEGF165 was highly expressed in MSG cells and was secreted into the cocoon of S1-V165; (2) the dimeric form of rhVEGF165 could be easily dissolved from S1-V165 cocoons using an alkaline solution; (3) rhVEGF165 extracted from S1-V165 cocoons exhibited slightly better cell proliferative activity than the hVEGF165 standard in cultured human umbilical vein endothelial cells. This study provides an alternative strategy for the production of bioactive rhVEGF165 using the MSG of transgenic silkworms.

Keywords

Silkworm Transgene Middle silk gland Recombinant human VEGF165 Bioactivity 

Notes

Acknowledgements

We apologize to our colleagues whose work was not cited in this paper due to the publisher’s space constraints. This work was supported by Grants (31872291, 31801126) from the National Natural Science Foundation of China and the Chongqing Research Program of Basic Research and Frontier Technology (cstc2017jcyjBX0041, cstc2017jcyj-yszx0009). The English in the manuscript was polished by American Journal Experts.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interests.

References

  1. Bencúrová M, Hemmer W, Focke-Tejkl M, Wilson IB, Altmann F (2004) Specificity of IgG and IgE antibodies against plant and insect glycoprotein glycans determined with artificial glycoforms of human transferrin. Glycobiology 14(5):457–466PubMedGoogle Scholar
  2. Byun J, Heard JM, Huh JE, Park SJ, Jung EA, Jeong JO, Gwon HC, Kim DK (2001) Efficient expression of the vascular endothelial growth factor gene in vitro and in vivo, using an adeno-associated virus vector. J Mol Cell Cardiol 33:295–305PubMedGoogle Scholar
  3. Cohen T, Gitay-Goren H, Neufeld G, Levi BZ (1992) High levels of biologically active vascular endothelial growth factor (VEGF) are produced by the baculovirus expression system. Growth Factors 7:131–138PubMedGoogle Scholar
  4. Connolly DT, Olander JV, Heuvelman D, Nelson R, Monsell R, Siegel N, Haymore BL, Leimgruber R, Feder J (1989) Human vascular permeability factor. Isolation from U937 cells. J Biol Chem 264:20017–20024PubMedGoogle Scholar
  5. Dehghanian F, Hojati Z (2014) Comparative insight into expression of recombinant human VEGF111b, a newly identified anti-angiogenic isoform, in eukaryotic cell lines. Gene 553:57–62PubMedGoogle Scholar
  6. Ferrara N (1999) Molecular and biological properties of vascular endothelial growth factor. J Mol Med 77:527–543PubMedGoogle Scholar
  7. Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O’Shea KS, Powell-Braxton L, Hillan KJ, Moore MW (1996) Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 380:439–442PubMedGoogle Scholar
  8. Ferrara N, Gerber HP, Lecouter J (2003) The biology of VEGF and its receptors. Nat Med 9:669–676Google Scholar
  9. Fiebich L, Jager B, Schollmann C, Weindel K, Wilting J, Kochs G, Marme D, Hug H (1993) Synthesis and assembly of functionally active human vascular endothelial growth factor homodimers in insect cells. Eur J Biochem 211:19–26PubMedGoogle Scholar
  10. Gupta R, Tongers J, Losordo DW (2009) Human studies of angiogenic gene therapy. Circ Res 105:724–736PubMedPubMedCentralGoogle Scholar
  11. Hoeben A, Landuyt B, Highley MS, Wildiers H, Van Oosterom AT, De Bruijn EA (2004) Vascular endothelial growth factor and angiogenesis. Pharmacol Rev 56:549–580PubMedGoogle Scholar
  12. Holmes K, Roberts OL, Thomas AM, Cross MJ (2007) Vascular endothelial growth factor receptor-2: structure, function, intracellular signalling and therapeutic inhibition. Cell Signal 19:2003–2012PubMedGoogle Scholar
  13. Horn C, Wimmer EA (2000) A versatile vector set for animal transgenesis. Dev Genes Evol 210:630–637PubMedGoogle Scholar
  14. Iizuka M, Ogawa S, Takeuchi A, Nakakita S, Kubo Y, Miyawaki Y, Hirabayashi J, Tomita M (2009) Production of a recombinant mouse monoclonal antibody in transgenic silkworm cocoons. FEBS J 276(20):5806–5820PubMedGoogle Scholar
  15. Itoh K, Kobayashi I, Nishioka S, Sezutsu H, Machii H, Tamura T (2016) Recent progress in development of transgenic silkworms overexpressing recombinant human proteins with therapeutic potential in silk glands. Drug Discov Ther 10:34–39PubMedGoogle Scholar
  16. Kang WK, Lee MH, Kim YH, Kim MY, Kim JY (2013a) Enhanced secretion of biologically active, non-glycosylated VEGF from Saccharomyces cerevisiae. J Biotechnol 164:441–448PubMedGoogle Scholar
  17. Kang W, Kim S, Lee S, Jeon E, Lee Y, Yun YR, Suh CK, Kim HW, Jang JH (2013b) Characterization and optimization of vascular endothelial growth factor (165) (rhVEGF(165)) expression in Escherichia coli. Protein Expr Purif 87(2):55–60PubMedGoogle Scholar
  18. Lee GY, Jung WW, Kang CS, Bang IS (2006) Expression and characterization of human vascular endothelial growth factor (VEGF165) in insect cells. Protein Expr Purif 46:503–509PubMedGoogle Scholar
  19. Lee SB, Park JS, Lee S, Park J, Yu S, Kim H, Kim D, Byun TH, Baek K, Ahn YJ, Yoon J (2008) Overproduction of recombinant human VEGF (vascular endothelial growth factor) in Chinese hamster ovary cells. J Microbiol Biotechnol 18:183–187PubMedGoogle Scholar
  20. Mitra N, Sinha S, Ramya TN, Surolia A (2006) N-linked oligosaccharides as outfitters for glycoprotein folding, form and function. Trends Biochem Sci 31(3):156–163PubMedGoogle Scholar
  21. Mohanraj D, Olson T, Ramakrishnan S (1995) Expression of biologically active human vascular endothelial growth factor in yeast. Growth Factors 12:17–27PubMedGoogle Scholar
  22. Morse MA (2001) Technology evaluation: VEGF165 gene therapy, Valentis Inc. Curr Opin Mol Ther 3:97–101PubMedGoogle Scholar
  23. Nguyen MT, Krupa M, Koo BK, Song JA, Vu TT, Do BH, Nguyen AN, Seo T, Yoo J, Jeong B, Jin J, Lee KJ, Oh HB, Choe H (2016) Prokaryotic soluble overexpression and purification of human VEGF165 by fusion to a maltose binding protein tag. PLoS ONE 11(5):e0156296PubMedPubMedCentralGoogle Scholar
  24. 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 128:531–544PubMedGoogle Scholar
  25. Park JS, Seo HS, Yum JS, Moon HM, Lee J (2005) The influence of N-glycosylation and C-terminal sequence on secretion of HBV large surface antigen from S. cerevisiae. Biotechnol Bioeng 92(2):250–255PubMedGoogle Scholar
  26. Peretz D, Gitay-Goren H, Safran M, Kimmel N, Gospodarowicz D, Neufeld G (1992) Glycosylation of vascular endothelial growth factor is not required for its mitogenic activity. Biochem Biophys Res Commun 182(3):1340–1347PubMedGoogle Scholar
  27. Poltorak Z, Cohen T, Sivan R, Kandelis Y, Spira G, Vlodavsky I, Keshet E, Neufeld G (1997) VEGF145, a secreted vascular endothelial growth factor isoform that binds to extracellular matrix. J Biol Chem 272:7151–7158PubMedGoogle Scholar
  28. Samuel RVM, Farrukh SY, Rehmat S, Hanif MU, Ahmed SS, Musharraf SG, Durrani FG, Saleem M, Gul R (2018) Soluble production of human recombinant VEGF-A121 by using SUMO fusion technology in Escherichia coli. Mol Biotechnol 60:585–594PubMedGoogle Scholar
  29. Santulli G (ed) (2013) Angiogenesis: insights from a systematic overview. Nova Science, New York. ISBN 978-1-62618-114-4Google Scholar
  30. Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS, Dvorak HF (1983) Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 219:983–985PubMedGoogle Scholar
  31. Senger DR, Perruzzi CA, Feder J, Dvorak HF (1986) A highly conserved vascular permeability factor secreted by a variety of human and rodent tumor cell lines. Cancer Res 46:5629–5632PubMedGoogle Scholar
  32. Siemeister G, Schnurr B, Mohrs K, Schachtele C, Marme D (1996) Expression of biologically active isoforms of the tumor angiogenesis factor VEGF in Escherichia coli. Biochem Biophys Res Commun 222:249–255PubMedGoogle Scholar
  33. Stephan CC, Brock TA (1996) Vascular endothelial growth factor, a multifunctional polypeptide. P R Health Sci J 15:169–178PubMedGoogle Scholar
  34. Taimeh Z, Loughran J, Birks EJ, Bolli R (2013) Vascular endothelial growth factor in heart failure. Nat Rev Cardiol 10:519–530PubMedGoogle Scholar
  35. Takahashi H, Shibuya M (2005) The vascular endothelial growth factor (VEGF)/VEGF receptor system and its role under physiological and pathological conditions. Clin Sci 109:227–241PubMedGoogle Scholar
  36. Taktak-BenAmar A, Morjen M, Ben Mabrouk H, Abdelmaksoud-Dammak R, Guerfali M, Fourati-Masmoudi N, Marrakchi N, Gargouri A (2017) Expression, purification and functionality of bioactive recombinant human vascular endothelial growth factor VEGF165 in E. coli. AMB Express 7(1):33PubMedPubMedCentralGoogle Scholar
  37. Tischer E, Mitchell R, Hartman T, Silva M, Gospodarowicz D, Fiddes JC, Abraham JA (1991) The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. J Biol Chem 266:11947–11954PubMedGoogle Scholar
  38. Tokuda H, Kozawa O, Uematsu T (2000) Basic fibroblast growth factor stimulates vascular endothelial growth factor release in osteoblasts: divergent regulation by p42/p44 mitogen-activated protein kinase and p38 mitogen-activated protein kinase. J Bone Miner Res 15(12):2371–2379PubMedGoogle Scholar
  39. Tomita M (2011) Transgenic silkworms that weave recombinant proteins into silk cocoons. Biotechnol Lett 33(4):645–654PubMedGoogle Scholar
  40. Walter DH, Hink U, Asahara T, Van Belle E, Horowitz J, Tsurumi Y, Vandlen R, Heinsohn H, Keyt B, Ferrara N, Symes JF, Isner JM (1996) The in vivo bioactivity of vascular endothelial growth factor/vascular permeability factor is independent of N-linked glycosylation. Lab Invest 74(2):546–556PubMedGoogle Scholar
  41. Wang G, Xia Q, Cheng D, Duan J, Zhao P, Chen J, Zhu L (2008) Reference genes identified in the silkworm Bombyx mori during metamorphism based on oligonucleotide microarray and confirmed by qRT-PCR. Insect Sci 15(5):405–413Google Scholar
  42. 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 22:925–938PubMedGoogle Scholar
  43. Xu H (2014) The advances and perspectives of recombinant protein production in the silk gland of silkworm Bombyx mori. Transgenic Res 23:697–706PubMedGoogle Scholar
  44. Xu H, Yuan L, Wang F, Wang Y, Wang R, Song C, Xia Q, Zhao P (2014) Overexpression of recombinant infectious bursal disease virus (IBDV) capsid protein VP2 in the middle silk gland of transgenic silkworm. Transgenic Res 23:809–816PubMedGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.State Key Laboratory of Silkworm Genome Biology, College of BiotechnologySouthwest UniversityChongqingChina

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