J Korean Cancer Assoc.  2000 Jun;32(3):595-604.

Production of Soluble VEGF Receptor Mutants for Inhibition of Angiogenesis

• Yun SY
• Hong YK
• Lee Y
• Kim K
• Kim HK
• Joe YA

Abstract

PURPOSE: 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.
MATERIALS AND METHODS
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.
RESULTS
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.
CONCLUSION
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.

Keyword

Angiogenesis; Vascular endothelial growth factor; Baculovirus; FLT-1; Tyrosine kinase; Mutant receptor

MeSH Terms

Angiogenesis Inducing Agents
Baculoviridae
Cellulose
Chromatography, Agarose
Clone Cells
DNA, Complementary
Heparin
Humans
Inclusion Bodies
Insects
Membranes
Mutant Proteins
Neoplasm Metastasis
Protein-Tyrosine Kinases
Receptors, Vascular Endothelial Growth Factor*
RNA
Umbilical Veins
Vascular Endothelial Growth Factor A*
Angiogenesis Inducing Agents
Cellulose
DNA, Complementary
Heparin
Mutant Proteins
Protein-Tyrosine Kinases
RNA
Receptors, Vascular Endothelial Growth Factor
Vascular Endothelial Growth Factor A