Targeting expression of the catalytic domain of the kinase insert domain receptor (KDR) in the peroxisomes of Pichia pastoris.
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.
Wang
Ya
Y
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
Xuan
Yaoji
Y
Zhang
Ping
P
Jiang
Xi
X
Ni
Zhenhua
Z
Tong
Linjiang
L
Zhou
Xiangshan
X
Lin
Liping
L
Ding
Jian
J
Zhang
Yuanxing
Y
D020134:Catalytic Domain; D017930:Genes, Reporter; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D020675:Peroxisomes / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D040301:Vascular Endothelial Growth Factor Receptor-2