文献类型：期刊文献

英文题名：Targeting expression of the catalytic domain of the kinase insert domain receptor (KDR) in the peroxisomes of Pichia pastoris

作者：Wang, Ya[1];Xuan, Yaoji[1];Zhang, Ping[1];Jiang, Xi[1];Ni, Zhenhua[1];Tong, Linjiang[2];Zhou, Xiangshan[1];Lin, Liping[2];Ding, Jian[2];Zhang, Yuanxing[1]

机构：[1]E China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Chinese Acad Sci, Shanghai Inst Mat Med, Div Antitumor Pharmacol, State Key Lab Drug Res, Shanghai 200031, Peoples R China

年份：2009

卷号：9

期号：5

起止页码：732

外文期刊名：FEMS YEAST RESEARCH

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000267751800006)】；

基金：We are grateful to Prof. Suresh Subramani, Section of Molecular Biology, University of California, San Diego, for the gift of plasmid pRDM054 and suggestive discussion. We also acknowledge Dr J.A.K.W. Kiel, Molecular Cell Biology, GBB, University of Groningen, the Netherlands, for suggestive discussion. This work was supported by The National Basic Research (973) Program of China (2004CB518903) and Shanghai Leading Academic Discipline Project ( Project Number: B505).

语种：英文

外文关键词：KDR; kinase insert domain receptor; peroxisome; phosphorylation; Pichia pastoris; vascular endothelial growth factor receptor 2

摘要：The kinase insert domain receptor (KDR), also known as vascular endothelial growth factor receptor-2 (VEGFR2), is an important therapeutic target for the treatment of cancer because of its crucial role in angiogenesis, which is fundamental to the malignancy of tumors. Here, we expressed the catalytic domain of KDR in Pichia pastoris under the control of the AOX1 promoter. In order to facilitate its purification and detection, His-tag and green fluorescent protein (GFP) were fused to the N-terminus of KDR. At the same time, a peroxisomal targeting signal 1 (SKL) was fused to the C-terminus to avoid the potential negative effect on the host cell. The highly expressing clone K1 was selected by GFP fluorescence intensity analysis using flow cytometry (FCM). Furthermore, the GFP-KDR-SKL fusion protein was proved to be correctly targeted to the peroxisomes of P. pastoris by colocation with blue fluorescent protein-SKL. The expression of GFP-KDR-SKL led to extensive phosphorylation of endogenous proteins and significantly inhibited cell growth. However, the expression was not lethal to the cells. Both in vitro biological activity assay and inhibition rate assay demonstrated that the purified GFP-KDR-SKL fusion protein exhibited high kinase catalytic activity and could be used as a target for anticancer drug screening.