Expression and characterization of a soluble VEGF receptor 2 protein

详细信息    查看全文

• 作者：Wei Liu (7) (8)
  Xinyuan Zhang (7)
  Ching Song (8)
  Shisan Bao (6)
  Donna Lai (7)
  Jianqiu Mou (8)
  Tao Jiang (8)
  Ningli Wang (7)
  
• 关键词：Gene transfection ; Truncated soluble protein ; Gene construction ; Transient transfection
• 刊名：Cell & Bioscience
• 出版年：2014
• 出版时间：December 2014
• 年：2014
• 卷：4
• 期：1
• 全文大小：921 KB
• 参考文献：1. Senger DR, Connolly DT, Van de Water L, Feder J, Dvorak HF: Purification and NH2-terminal amino acid sequence of guinea pig tumor-secreted vascular permeability factor. / Cancer Res 1990, 50:1774-778.
  2. Cooper ME, Vranes D, Youssef S, Stacker SA, Cox AJ, Rizkalla B, Casley DJ, Bach LA, Kelly DJ, Gilbert RE: Increased renal expression of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 in experimental diabetes. / Diabetes 1999, 48:2229-239. CrossRef
  3. Zhang X, Chen M, Gillies MC: Two isoforms of Flk-1 transcripts in early diabetic rat retinas. / Curr Eye Res 2012, 37:73-9. CrossRef
  4. Ferrara N, Gerber HP, LeCouter J: The biology of VEGF and its receptors. / Nat Med 2003, 9:669-76. CrossRef
  5. Stuttfeld E, Ballmer-Hofer K: Structure and function of VEGF receptors. / IUBMB Life 2009, 61:915-22. CrossRef
  6. Jacobi J, Tam BY, Wu G, Hoffman J, Cooke JP, Kuo CJ: Adenoviral gene transfer with soluble vascular endothelial growth factor receptors impairs angiogenesis and perfusion in a murine model of hindlimb ischemia. / Circulation 2004, 110:2424-429. CrossRef
  7. Kou B, Li Y, Zhang L, Zhu G, Wang X, Li Y, Xia J, Shi Y: In vivo inhibition of tumor angiogenesis by a soluble VEGFR-2 fragment. / Exp Mol Pathol 2004, 76:129-37. CrossRef
  8. Zhang X, Bao S, Hambly BD, Gillies MC: Vascular endothelial growth factor-A: a multifunctional molecular player in diabetic retinopathy. / Int J Biochem Cell Biol 2009, 41:2368-371. CrossRef
  9. Matthews W, Jordan CT, Gavin M, Jenkins NA, Copeland NG, Lemischka IR: A receptor tyrosine kinase cDNA isolated from a population of enriched primitive hematopoietic cells and exhibiting close genetic linkage to c-kit. / Proc Natl Acad Sci USA 1991, 88:9026-030. CrossRef
  10. Roskoski R Jr: VEGF receptor protein-tyrosine kinases: structure and regulation. / Biochem Biophys Res Commun 2008, 375:287-91. CrossRef
  11. Li H, Cao W, Chen Z, Acheampong DO, Jin H, Li D, Zhang J, Wang M: The antiangiogenic activity of a soluble fragment of the VEGFR extracellular domain. / Biomed Pharmacother 2013, 67:599-06. CrossRef
  12. Leopold AV, Baklaushev VP, Pavlov KA, Chekhonin VP: Expression of a recombinant extracellular fragment of human vascular endothelial growth factor receptor VEGFR1 in E. coli. / Bull Exp Biol Med 2011, 151:347-52. CrossRef
  13. Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z: Vascular endothelial growth factor (VEGF) and its receptors. / The FASEB Journal 1999, 13:9-2.
  14. Farnia P, Bandehpour M, Ghanavi J, Kazemi B: Cloning and expression of soluble vascular endothelial growth factors receptor-1 (sFlt-1) fragments in CHO-K1. / Int J Clin Exp Med 2013, 6:773-78.
  15. Baldi L, Muller N, Picasso S, Jacquet R, Girard P, Thanh HP, Derow E, Wurm FM: Transient gene expression in suspension HEK-293 cells: application to large-scale protein production. / Biotechnol Prog 2005, 21:148-53. CrossRef
  
• 作者单位：Wei Liu (7) (8)
  Xinyuan Zhang (7)
  Ching Song (8)
  Shisan Bao (6)
  Donna Lai (7)
  Jianqiu Mou (8)
  Tao Jiang (8)
  Ningli Wang (7)
  
  7. Bosch Institute, F13, the University of Sydney, Sydney, NSW, 2006, Australia
  8. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology (IGDB), No.1 West Beichen Road, Chaoyang District, Beijing, 100101, China
  6. Discipline of Pathology, D06, Bosch Institute, the University of Sydney, Sydney, NSW, 2006, Australia
  
• ISSN：2045-3701

文摘

Objective To clone and express a truncated, soluble vascular endothelial growth factor receptor 2 (sVEGFR2) possessing the combined-functional domains 1- and 5 in eukaryotic cells and to test the inhibitory effects of full length VEGFR2 in vivo. Results pCMV6-trunctated-rVegfr2 (6100 bp) was successfully cloned. The transfection experiments showed that either pCMV6-truncated-rat-Vegfr2 (pCMV6-truncated-rVegfr2) or pCMV6-rVegfr2 inhibited the expression of intracellular green fluorescent protein, which is usually used as an exogenous transfected reporter gene to determine the transfected efficiency. An analysis of the transfected cells revealed that the amount of full-length VEGFR2 protein in the pCMV6-truncated-rVegfr2 transfected cells was 20% lower than that in the negative control (non-transfected HEK 293 cells). The differences in test results between the transfected and negative control groups were greatest from 24-0 h after transfection; this period was therefore chosen as optimal for collecting culture supernatants. This analysis was highly sensitive for detecting the amount of sVEGFR2 protein expressed and secreted by the cells, and the sVEGFR2 protein content was found to increase by approximately 26% in the transfected cells compared to that in the negative control cells (68.2% ± 1.7% vs. 41.9% ± 2.9%, P = 0.000) and by 18% compared to the negative control cells (68.2% ± 1.7% vs. 50.0% ± 0.5%, P = 0.003). Propidium iodide and Hoechst staining indicated no significant change in the number of HEK293 cells undergoing apoptosis 6 days after pCMV6-trucated-Vegfr2 transfection, compared to the negative control. Soluble VEGFR2 produced by pCMV6-truncated-rVegfr2 inhibited full-length VEGFR2 protein expression in the cell membrane. Conclusions This study employed a eukaryotic system to express sVEGFR2. The use of transient transfection technology greatly improved transfect efficiency. Recombinant sVEGFR2 inhibited the effect of endogenous full-length VEGFR2 but was not cytotoxic.