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Calmodulin as a Versatile Tag for Antibody Fragments

Bio/Technology volume 13, pages 373–377 (1995)Cite this article

Abstract

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 > 109 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-l-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.

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Author notes

  1. Dario Neri: Corresponding author.

Authors and Affiliations

  1. Cambridge Centre for Protein Engineering, MRC Centre, Hills Road, Cambridge, CB2 2QH, UK

    Dario Neri, Claudia de Lalla, Heike Petrul & Greg Winter

  2. DIBIT, San Raffaele Scientific Institute, Via Olgettina 58, 20132, Milano, Italy

    Claudia de Lalla

  3. Departimento di Biologia Molecolare, Universita' di Siena, Centro Didattico loc. Le Scotte, 53100, Siena, Italy

    Paolo Neri

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Neri, D., Lalla, C., Petrul, H. et al. Calmodulin as a Versatile Tag for Antibody Fragments. Nat Biotechnol 13, 373–377 (1995). https://doi.org/10.1038/nbt0495-373

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