Abstract
Intracellular localization is important for the characterization of a gene product. Microscopy of fluorescent protein fusions has become the method of choice to define the spatial and temporal behavior of a protein. We show here that recombinant antibody fluorescent protein fusions can be used to monitor the localization of intracellular antigens in fixed or living cells. A most successful application of phage-display technology has been the isolation of recombinant antibodies from large combinatorial repertoires. The most versatile antibody format is the single-chain Fv fragment (scFv) in which a flexible polypeptide linker joins the heavy- and light-chain antibody variable domains. Commercial systems are now available to produce scFv phage-display libraries encoding a large pool of binding specificities from which antibodies can be isolated and used as immunochemical or intracellular reagents. We designed a plasmid for ectopic expression of a recombinant antibody fused to a green fluorescent protein (GFP) under the control of an attenuated nmt1 promoter in Schizosaccharomyces pombe. The antibody binds to its target antigen without inhibiting protein function, allowing visualization of its intracellular location in fixed or living cells.
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© 2006 Humana Press Inc., Totowa, NJ
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Alting-Mees, M.A., Risseeuw, E.P., Liu, E., Desautels, M., Crosby, W.A., Hemmingsen, S.M. (2006). Intracellular Expression of Recombinant Antibody Fluorescent Protein Fusions for Localization of Target Antigens in Schizosaccharomyces pombe . In: Xiao, W. (eds) Yeast Protocol. Methods in Molecular Biology, vol 313. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-958-3:097
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DOI: https://doi.org/10.1385/1-59259-958-3:097
Publisher Name: Humana Press, Totowa, NJ
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