Fluobodies: green fluorescent single-chain Fv fusion proteins.
An expression system (pSKGFP), which permits the expression of single-chain variable fragments as fusion proteins with modified green fluorescent proteins, was designed. This expression system is comparable to frequently used phage display vectors and allows single-step characterization of the selected recombinant antibodies by flow cytometry or fluorescent cell staining. Two different single-chain variable fragment antibodies, both directed against the lipopolysaccharide of the bacterium Ralstonia solanacearum have been genetically fused to a red-shifted green fluorescent protein and the produced fusion protein tested for usefulness. These fluobodies can be produced in cultures of bacterial cells and purified using immobilized metal affinity chromatography. They function well in flow cytometry and immunofluorescent cell staining, are specific for their target antigens and, unlike FITC-conjugated antibodies, they do not fade upon illumination.
Griep
R A
RA
Laboratory of Monoclonal Antibodies, Wageningen University and Research Centre, P.O. Box 8123, 6700 ES, Wageningen, The Netherlands.
van Twisk
C
C
van der Wolf
J M
JM
Schots
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D019117:Burkholderia / Q000276:immunology; D017931:DNA Primers / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D005434:Flow Cytometry; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D008070:Lipopolysaccharides / Q000276:immunology; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology
D017931:DNA Primers; D004274:DNA, Recombinant; D007128:Immunoglobulin Fragments; D008070:Lipopolysaccharides; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv; D049452:Green Fluorescent Proteins