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Enzyme Microb Technol. 2014 Sep;63:58-63. doi: 10.1016/j.enzmictec.2014.05.007. Epub 2014 Jun 7.

Production of functional human vascular endothelial growth factor(165) in transgenic rice cell suspension cultures.

Author information

  • 1Department of Molecular Biology, Chonbuk National University, Dukjindong 664-14, Jeonju, Jeollabuk-do 561-756, Republic of Korea; Department of Bioactive Material Sciences, Chonbuk National University, Dukjindong 664-14, Jeonju, Jeollabuk-do 561-756, Republic of Korea.
  • 2Department of Molecular Biology and the Institute for Molecular Biology and Genetics, Chonbuk National University, Jeonju 561-756, Republic of Korea.
  • 3Department of Molecular Biology, Chonbuk National University, Dukjindong 664-14, Jeonju, Jeollabuk-do 561-756, Republic of Korea; Department of Bioactive Material Sciences, Chonbuk National University, Dukjindong 664-14, Jeonju, Jeollabuk-do 561-756, Republic of Korea. Electronic address: mskyang@jbnu.ac.kr.

Abstract

Vascular endothelial growth factors (VEGFs) are secreted by tumor cells and other cells exposed to hypoxia, and play a critical role in the development and differentiation of the vascular system. In this study, we investigated the production of functional recombinant human VEGF165 (rhVEGF165) in transgenic rice cell suspension culture. Complementary DNA was synthesized from human leukemia HL60 cells and cloned into expression vectors under the control of the rice α-amylase 3D (RAmy3D) promoter. The rice seed (Oryza sativa L. cv. Dongjin) was transformed with this recombinant vector by the Agrobacterium mediated method and the integration of the target gene into the plant genome was confirmed by genomic PCR. The expression of rhVEGF165 in the rice cells was determined by Northern blot and Western blot analyses. The accumulated rhVEGF165 protein in the culture medium was 19 mg/L after 18 days of culturing in a sugar-free medium. The rhVEGF165 was purified using a heparin HP column and its biological activity was tested on human umbilical vein endothelial cells (HUVECs). The purified rhVEGF165 significantly increased the proliferative activity of the HUVECs. Therefore, it was demonstrated that functional rhVEGF165 could be produced using transgenic rice suspension culture vector under the control of the RAmy3D promoter.

Copyright © 2014 Elsevier Inc. All rights reserved.

KEYWORDS:

Heparin HP column; RAmy3D promoter; Recombinant human VEGF(165); Rice cell suspension culture

PMID:
25039061
[PubMed - indexed for MEDLINE]
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