Bacterial production of integrin αvβ3 targeting optical molecular imaging probe

1594 Objectives Aims of this study is to establish a bacterial clone which secretes αvβ3 targeting probe containing bioluminescence and fluorescence activities,and to verify its specific targeting and optical activities using molecular imaging technique. Methods We constructed a bacterial expression vector which produces secretory gaussia luciferase(sGluc),mCherry and RGD as a fusion protein(sGluc-mCherry-RGDX3; GCR)and a control vector which produces sGluc and mCherry as a fusion protein(sGluc-mCherry called GC). The genes of interest were cloned into pRSET(B) vector for His-tag purification and the pRSET(B) vector was transformed into the E. coli JM109 bacteria for GCR and GC production.The His-tagged recombinant proteins were purified using Ni-NTA resin column. Luciferase activity of the purified GCR and GC proteins were determined by luciferase assay and IVIS imaging in in vitro.Specific binding of GCR to the αvβ3 was evaluated with bioluminescene assay and confocal microscopy using αvβ3 expressing U87MG and CHO cells. Results The GCR and GC were expressed in the E coli and were secreted into growth medium. Luciferase activity of growth medium was about 10 fold higher than that of bacterial lysate. The GCR and GC proteins were successfully purified with His-tag purification method.The GCR protein showed higher binding affinity to the U87MG cells on confocal microscopy and IVIS imaging.The binding affinity of The GCR protein to U87MG cells was about 3 fold higher than to CHO cells. Conclusions We established E. coli clone which secretes specifically αvβ3 targeting optical molecular imaging probe containing bioluminescence and fluorescence activities.The molecular imaging protein probe might be produced inexpensively and feasibly used to evaluate neoangiogenesis in in vitro and in vivo study