Growth Factor (hnTf-VEGF) Fusion Protein Containing an Integrated Binding Site for 111In for Imaging Tumor Angiogenesis

Our objective was to synthesize a recombinant protein (hnTf-VEGF [VEGF is vascular endothelial growth factor]) composed of VEGF165 fused through a flexible polypeptide linker (GGGGS)3 to the n-lobe of human transferrin (hnTf) for imaging angiogen-esis. The hnTf domain allowed labeling with 111In at a site remote from the VEGF receptor-binding domain. Methods: DNA en-coding hnTf, peptide linker (GGGGS)3, and VEGF165 genes were cloned into the Pichia pastoris vector pPICZB to generate the pPICZB-hnTF-VEGF plasmid. The expression vector was transformed into P. pastoris KM71H strain. The protein was purified using Co2 metal affinity resin. The growth-stimulatory effects of hnTf-VEGF on human umbilical vascular endothelial cells (HUVECs) and its binding to porcine aortic endothelial cells (PAECs) transfected with VEGF receptors were evaluated. hnTf-VEGF protein was labeled with 111InCl3 in 10 mmol/L HEPES/1