Production of functional human vascular endothelial growth factor165 in transgenic rice cell suspension cultures

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 VEGF(165) (rhVEGF(165)) 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 a-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 rhVEGF(165) in the rice cells was determined by Northern blot and Western blot analyses. The accumulated rhVEGF(165) protein in the culture medium was 19 mg/L after 18 days of culturing in a sugar-free medium. The rhVEGF(165) was purified using a heparin HP column and its biological activity was tested on human umbilical vein endothelial cells (HUVECs). The purified rhVEGF(165) significantly increased the proliferative activity of the HUVECs. Therefore, it was demonstrated that functional rhVEGF(165) could be produced using transgenic rice suspension culture vector under the control of the RAmy3D promoter. (C) 2014 Elsevier Inc. All rights reserved.