Intracellular Expression of Recombinant Antibody Fluorescent Protein Fusions for Localization of Target Antigens in Schizosaccharomyces pombe

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.