Design of metal-binding green fluorescent protein variants

摘要

Diglycylcysteine motifs bind reduced oxo-compounds of technetium-99m, an important isotope in nuclear imaging. We suggested a system for detecting gene expression employing the effect of oxo[~(99m)Tc]technetate (Tc(V)O~(3+)) transchelation and coordination with redox amino acid motifs. DNA fragments encoding diglycylcysteine (GGC) binding motifs were prepared by PCR and positioned downstream from the green fluorescent protein (GFP) cDNA insert. Using a Bluescript (+) vector with the fusion protein positioned under the control of a lac promoter, we obtained several E. coli clones expressing the following GFP fusion peptides: (1) GFP-P1 bearing a 'hydrophilic' C-terminal peptide (LEGGGCEGGC) containing two residues of glutamic acid and C-terminal cysteine (2) GFP-P2 carrying a 'hydrophobic' (LGGGGCGGGCGI) peptide (3) a control GFP fusion peptide with deleted C-terminal portion. Bacterial lysates obtained from the corresponding clones were tested for oxo[~(99m)Tc]technetate transchelation from a glucoheptonate complex. We found, using a solid phase assay, that radioactivity associated with protein lysates obtained from clones expressing GFP-P2 fusions were 3-4 fold higher than lysates prepared from a clone expressing a truncated GFP fusion protein lacking the C-terminal GGC motifs. High expression of GFP fusions (5-21% of total protein) was demonstrated by electrophoresis and verified by immunoblotting. Specific association of the isotope with GFP-P2 fusion proteins was detected upon incubation of gels in the presence of [~(99m)Tc]glucoheptonate, while no binding of oxo[~(99mTc)]technetate to GFP-P1 was revealed. We demonstrated, by using semi-quantitative autoradiography, that there is a 10-fold higher binding of oxotechnetate to GFP-P2 than to a control GFP fusion protein. The implications of the study for in vivo gene expression imaging are discussed.