DESIGN OF METAL-BINDING GREEN FLUORESCENT PROTEIN VARIANTS

Diglycylcysteine motifs bind reduced oxo-compounds of technetium-99m, an important isotope in nuclear imaging. We suggested a system for det ecting gene expression employing the effect of oxo[Tc-99m]technetate ( Tc(V)O3+) transchelation and coordination with redox amino acid motifs . DNA fragments encoding diglycylcysteine (GGC) binding motifs were pr epared by PCR and positioned downstream from the green fluorescent pro tein (GFP) cDNA insert. Using a Bluescript (+) vector with the fusion protein positioned under the control of a lac promoter, we obtained se veral E. coli clones expressing the following GFP fusion peptides: (1) GFP-P1 bearing a 'hydrophilic' C-terminal peptide (LEGGGCEGGC) contai ning two residues of glutamic acid and C-terminal cysteine (2) GFP-P2 carrying a 'hydrophobic' (LGGGGCGG-GCGI) peptide (3) a control GFP fus ion peptide with deleted C-terminal portion. Bacterial lysates obtaine d from the corresponding clones were tested for oxo[Tc-99m]technetate transchelation from a glucoheptonate complex. We found, using a solid phase assay, that radioactivity associated with protein lysates obtain ed from clones expressing GFP-P2 fusions were 3-4 fold higher than lys ates prepared from a clone expressing a truncated GFP fusion protein l acking the C-terminal GGC motifs. High expression of GFP fusions (5-21 % of total protein) was demonstrated by electrophoresis and verified b y immunoblotting. Specific association of the isotope with GFP-P2 fusi on proteins was detected upon incubation of gels in the presence of [T c-99m]glucoheptonate, while no binding of oxo[Tc-99m]technetate to GFP -P1 was revealed. We demonstrated, by using semi-quantitative autoradi ography, that there is a 10-fold higher binding of oxotechnetate to GF P-P2 than to a control GFP fusion protein. The implications of the stu dy for in vivo gene expression imaging are discussed. (C) 1998 Elsevie r Science B.V.