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Novel Approaches to Molecular ImagingOptical and Bioluminescence |
1 Dept of Radiology, Stanford University, Stanford, California
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265
Objectives: VEGF121 loses its receptor affinity after chemically modified with bulky molecules such as fluorescent dyes. To remedy this, we produced VEGF121-Avi fusion protein, which can be site-specifically biotinylated at K residue within the Avi-tag by BirA enzyme. The resulting site-specifically biotinylated VEGF121-Avi (VEGF-Avi-b) was characterized in vitro and its optical imaging properties was evaluated by using VEGF-Avi-b-streptavidin-IRDye800 (SA800) complex (abbreviated as VEGF-SA800) in tumor-bearing mice.
Methods: Recombinant VEGF121-Avi was expressed and purified. The binding affinity of VEGF121-Avi was evaluated by receptor binding assay. The site-specific biotinylaion was performed using BirA enzyme, and confirmed by western blot and mass mapping. The formation of VEGF-SA800 was confirmed by cell staining, SDS-PAGE and western blot. VEGF-SA800 was injected into 67NR-tumor bearing mice and its tumor targeting efficacy evaluated by CRI Maestro imaging system.
Results: The VEGF121-Avi was successfully expressed and purified. The binding affinity of VEGF121-Avi to PAE/KDR cells was 3.12±0.54 nM, which was comparable to that of wild-type VEGF121. VEGF121-Avi was site-specifically biotinylated by BirA as assessed by western blot and mass mapping. VEGF-Avi-b was able to form stable complex with SA800. As shown in Figure 1, VEGF-SA800 and SA800 has comparable tumor uptake at early time points but the difference becomes very obvious at 48 h p.i.
Conclusions: Site-specifically biotinylated VEGF121-Avi is a novel molecule well-suited for streptavidin binding and VEGFR targeted imaging.
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