Please use this identifier to cite or link to this item: http://hdl.handle.net/123456789/8942
Title: Periplasmic expression optimization of VEGFR2 D3 adopting response surface methodology: Antiangiogenic activity study
Authors: Cao, Wanlu
Li, Haixin
Zhang, Juan
Li, Daojuan
Acheampong, Desmond Omane
Chen, Zhiguo
Wang, Min
Keywords: Response surface methodology
Periplasmic expression
VEGFR2 D3
Antiangiogenic activity
VEGF blocker
Issue Date: 13-May-2013
Publisher: Elsevier
Abstract: Vascular endothelial growth factor (VEGF) is one of the most significant mediators of angiogenesis, which interacts with a specific membrane receptor: VEGF receptor 2 (VEGFR2). Studies elsewhere have shown that, a VEGF-blocker can regulate several vital processes of tumor promotion. However, there is no liter- ature evidence of investigation on antiangiogenic ability of single domain 3 of VEGFR-2 (VEGFR2 D3), as the key domain in signal transduction of VEGF. In this article, we aimed at developing an efficient method for producing soluble form of this receptor as therapeutic applications. The optimization of the produc- tion of soluble VEGFR2 D3 in Escherichia coli was firstly done by testing the periplasmic expression in dif- ferent expression systems using three osmotic shock methods. To enhance the yield, vital factors were selected from nine factors by Plackett–Burman design and the level of each viral factor was optimized via a response surface methodology based central composite design. After purification and identification of the protein, the bioactivity assays: quantitative ELISA, VEGF-induced proliferation and in vivo chick chorioallantoic membrane assay were employed in our study. The outcome showed that, E. coli Rosetta-gami (DE3)/pET22b-VEGFR2 D3 was the most effective expression system. Furthermore, the inducing time, peptone and glycerol concentration affected the periplasmic expression of VEGFR2 D3 sig- nificantly. The corresponding level was also optimized. The bioactivity assay studies showed VEGFR2 D3 could suppress both VEGF stimulated cell proliferation in vitro and neovascularization in vivo. We have therefore provided a novel antiangiogenic drug candidate relating to VEGF-VEGFR2 pathway.
URI: http://hdl.handle.net/123456789/8942
Appears in Collections:School of Allied Health Sciences

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