Bifunctional fusion proteins consisting of a single-chain antibody and an engineered lanthanide-binding protein.
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 Tb3+ with very high affinity and to give strong, sensitized Tb3+ 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 Tb3+ binding by a series of metal chelators indicated that the affinities of the oncomodulin and 30 residue fusions for Tb3+ 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.
MacKenzie
C R
CR
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada.
Clark
I D
ID
Evans
S V
SV
Hill
I E
IE
MacManus
J P
JP
Dubuc
G
G
Bundle
D R
DR
Narang
S A
SA
Young
N M
NM
Szabo
A G
AG
D000595:Amino Acid Sequence; D001483:Base Sequence; D001667:Binding, Competitive; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000276:immunology; D002352:Carrier Proteins / Q000138:chemical synthesis* / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics; D008163:Luminescent Measurements; D008674:Metals, Rare Earth / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D013725:Terbium / Q000378:metabolism
D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D008674:Metals, Rare Earth; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv; C034733:oncomodulin; D013725:Terbium