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J Korean Cancer Assoc > Volume 32(3); 2000 > Article
Journal of the Korean Cancer Association 2000;32(3): 595-604.
Production of Soluble VEGF Receptor Mutants for Inhibition of Angiogenesis
Soo Young Yun, Yong Kil Hong, Yoon Lee, Kwangsei Kim, Hoon Kyo Kim, Young Ae Joe
1Cancer Research Institute, The Catholic Research Institutes of Medical Sciences.
2Department of Neurosurgery, The Catholic University of Korea, Seoul.
3Department of Internal Medicine, St. Vincent's Hospital, Suwon, Korea.
Vascular endothelial growth factor (VEGF) is a potent angiogenic factor of many solid tumors, promoting vascularization and formation of metastases. In an attempt to generate effective VEGF inhibitors, the authors constructed the VEGF receptor mutants, expressed in E. coli and Sf9 insect cells, and examined their binding to VEGF.
The cDNA fragment encoding FLT-1 extracellular domain was cloned from human umbilical vein endothelial (HUVE) cell total RNA using RT-PCR. PCR- subcloning was performed using this template, in order to generate the deletion mutants by introducing FLT-1 partial sequences into E.coli expression vector pET-21d and baculovirus transfer vactors, pBAC-1 and pBAC-3. Two mutant proteins from baculovirus-infected insect cells were purified by heparin sepharose chromatography and immobilized into nitrdegrees Cellulose membrane followed by 125I-VEGF binding assay.
Two mutant receptors, sFLT (1~7) and sFLT (2~4) expressed in E.coli appeared in inclusion body as insoluble proteins. The soluble mutant receptors were produced in low yield by baculovirus/insect cell expression system. Both immobilized mutant receptors, sFLT (1~7) and sFLT (2~4) were able to bind VEGF.
These results suggest that a small soluble mutant receptor, sFLT (2~4), as well as sFLT (1~7) may be used effectively for bldegrees Cking angiogenic function of VEGF.
Key words: Angiogenesis;Vascular endothelial growth factor;Baculovirus;FLT-1;Tyrosine kinase;Mutant receptor
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