[Transient gene expression of soluble VEGFR2: I-IV].
The extracelluar domain I-IV of target gene VEGFR2 (Vascular endothelial growth factor receptor 2) was cloned from villus of trimester abortion by RT-PCR, and linked to the expression vectors. Then, the transfection conditions were optimized in serum-free suspension culture HEK293 using GFP (Green fluorescence protein) as the report gene. The results showed that the optimal transfection efficiency and cell number were obtained when the ratio of foreign DNA: PEI = 1:2 (W/W), DNA = 1.5 g /10(6) cells and shaking speed (120 r/min) in serum free medium in the beginning 4 hours of transfection. After optimizing the transfection conditions, the expression vector was successfully constructed for transient gene expression in HEK293, COS-7, and CHO-K1. The result shows that the target protein was only detected in CHO-K1 supernatant. Because of the C-terminal 8-His tag of target protein, target protein was subsequently purified using Ni2+-IDA and 5 mg purified protein was obtained in 1.5 L supernatant of CHOK1.
Li
Jun
J
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
Yi
Xiaoping
X
Zhang
Yuanxing
Y
Sun
Xiangming
X
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002824:Chorionic Villi / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D016895:Culture Media, Serum-Free; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012995:Solubility; D014162:Transfection; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000096:biosynthesis / Q000235:genetics*
D016895:Culture Media, Serum-Free; D012333:RNA, Messenger; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D040301:Vascular Endothelial Growth Factor Receptor-2