Cloning and Optimization of Soluble Vascular Endothelial Growth Factor(165) Expression in Escherichia coli

BACKGROUND: Vascular Endothelial Growth Factor (VEGF) is a coordinate regulator of physiological angiogenesis during embryogenesis, skeletal growth and reproductive functions. There are several types of VEGF, including VEGF(165). VEGFs stimulate endothelial cell growth, angiogenesis, and capillary permeability. Low induction temperature is a major factor for expression of the recombinant VEGF(165) in soluble form. The purpose of this study was cloning and optimization of soluble vascular endothelial growth factor(165) expression in Escherichia coli (E. coli). METHODS: In this study, total RNA of HeLa cell [cervix epithelium] was extracted. The VEGF(165) gene was amplified by reverse transcription polymerase chain reaction (RTPCR), and then VEGF(165) was subcloned into prokaryotic expression vectors pET-32a(+) and transformed into BL21 (DE3) E. coli strain. VEGF(165) expression was optimized by fine adjustments such as induction time and incubation temperature. VEGF(165) was analyzed by DNA sequencing prior to expression and the protein was further characterized by SDS-PAGE and immunoblotting using His•tag specific polyclonal antibody. RESULTS: Our results demonstrated that VEGF(165) was successfully cloned and expressed in pET-32a(+) vector. Optimization of the expression procedure showed that, induction by 1 mM IPTG at OD600=0.7 and overnight incubation at 22°C resulted in the highest expression levels of soluble VEGF(165). CONCLUSION: In this study, the expression of VEGF(165) in a high soluble level was successfully cloned and optimized.