Bifunctional fusion proteins consisting of a single-chain antibody and an engineered lanthanide-binding protein

Abstract The combination of an antibody fragment with a lanthanide chelating protein has desirable characteristics for fluorescence-based immunoassays and tumor radioimmunotherapy. As a model for this design, a fusion protein consisting of a single-chain antibody linked to an engineered version of oncomodulin, a protein with two Ca 2+ -binding motifs (the CD and EF loops), was produced by secretion from Escherichia coli in good yield. The single-chain antibody was specific for a Salmonella O -polysaccharide. The CD loop of oncomodulin had been redesigned to bind lanthanide ions with high affinity. The fusion protein was shown to have antigen-binding activity that was comparable to that of the unfused single-chain antibody, to bind Tb 3+ with very high affinity and to give strong, sensitized Tb 3+ luminescence via excitation of the tryptophan residue in the CD loop. A second fusion protein containing a 30-residue helix-loop-helix motif as the lanthanide-binding component was also prepared, but showed considerably lower solubility. Competition for Tb 3+ binding by a series of metal chelators indicated that the affinities of the oncomodulin and 30 residue fusions for Tb 3+ were approximately 10 11 M −1 and 10 7 M −1 , respectively. Time-resolved lanthanide luminescence photography of electrophoresis gels demonstrated that the helix-loop-helix Ca 2+ -binding could be used to specifically visualize the scFv fragment.