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.
Alting-Mees
Michelle A
MA
Therapeutics, Inc., Saskatoon, Saskatchewan, Canada.
Risseeuw
Eddy P
EP
Liu
Enwu
E
Desautels
Michel
M
Crosby
William A
WA
Hemmingsen
Sean M
SM
D000941:Antigens / Q000378:metabolism; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009172:Mycology / Q000379:methods; D019151:Peptide Library; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D014170:Transformation, Genetic
D000941:Antigens; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv; D049452:Green Fluorescent Proteins