Calmodulin as a versatile tag for antibody fragments.
Calmodulin is a highly acidic protein (net charge -24 at pH 8.0 in the absence of calcium) that binds to peptide and organic ligands with high affinity (Ka > 10(9) M-1) in a calcium-dependent manner. We have exploited these properties to develop calmodulin as a versatile tag for antibody fragments. Fusions of calmodulin with single chain Fv fragments (scFv) could be expressed by secretion from bacteria in good yield (5-15 mg/l in shaker flasks), and purified from periplasmic lysates or broth to homogeneity in a single step, either by binding to anion-exchange resin (DEAE-Sephadex), or to an organic ligand of calmodulin (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide-agarose). The antibody fusions could be detected by binding of fluorescently labeled peptide ligands, as illustrated by their use in confocal microscopy, fluorescent activated cell sorting and "band shift" gel electrophoresis. Moreover, the interaction between calmodulin and peptide ligands could provide a means of heterodimerization of proteins, as illustrated by the assembly of an antibody-calmodulin fusion with maltose binding protein tagged with a peptide ligand of calmodulin.
Neri
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D
Cambridge Centre for Protein Engineering, MRC Centre, UK.
de Lalla
C
C
Petrul
H
H
Neri
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P
Winter
G
G
D000595:Amino Acid Sequence; D018033:Antibodies, Bispecific / Q000737:chemistry / Q000235:genetics; D000911:Antibodies, Monoclonal / Q000235:genetics*; D001483:Base Sequence; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D003198:Computer Simulation; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes; D015870:Gene Expression; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000276:immunology; D011993:Recombinant Fusion Proteins
D018033:Antibodies, Bispecific; D000911:Antibodies, Monoclonal; D002147:Calmodulin; D005456:Fluorescent Dyes; D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; D009113:Muramidase