ABSTRACT
Structural information on intracellular fusions of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria with endogenous proteins is required as they are increasingly used in cell biology and biochemistry. We have investigated the dynamic properties of GFP alone and fused to a single chain antibody raised against lipopolysaccharide of the outer cell wall of gram-negative bacteria (abbreviated as scFv-GFP). The scFv moiety was functional as was proven in binding assays, which involved the use of both fluorescence correlation spectroscopy observing the binding of scFv-GFP to gram-negative bacteria and a surface plasmon resonance cell containing adsorbed lipopolysaccharide antigen. The rotational motion of scFv-GFP has been investigated with time-resolved fluorescence anisotropy. However, the rotational correlation time of scFv-GFP is too short to account for globular rotation of the whole protein. This result can only be explained by assuming a fast hinge motion between the two fused proteins. A modeled structure of scFv-GFP supports this observation.
Subject(s)
Immunoglobulin Variable Region/chemistry , Luminescent Proteins/chemistry , Animals , Cell Wall/immunology , Computer Graphics , Fluorescence Polarization , Gram-Negative Bacteria/immunology , Green Fluorescent Proteins , Lipopolysaccharides/immunology , Models, Molecular , Protein Conformation , Recombinant Fusion Proteins/chemistry , Scyphozoa , Single-Chain Antibodies , Spectrometry, FluorescenceABSTRACT
ABSTRACT A panel of recombinant single-chain antibodies (scFvs) against structural proteins of Tomato spotted wilt virus (TSWV) was retrieved from a human combinatorial scFv antibody library using the novel phage display technique. After subcloning the encoding DNA sequences in the expression vector pSKAP/S, which allowed the scFvs to be expressed as alkaline phosphatase fusion proteins, 17 different scFv antibodies were obtained. Of these, 12 scFvs were directed against the nucleoprotein (N) and 5, putatively, against the glycoproteins (G1 and G2). Five of the N-specific antibodies cross-reacted with two other tospoviruses (Tomato chlorotic spot virus and Groundnut ringspot virus), but none recognized the more distantly related tospoviruses Impatiens necrotic spot virus, Watermelon silverleaf mottle virus, Iris yellow spot virus, or Physalis severe mottle virus. The successful use of one of the antibodies as coating and detection reagent in a double-antibody sandwich enzyme-linked immunosorbent assay showed the potential of the phage display system in obtaining antibodies for routine TSWV diagnosis.
ABSTRACT
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.
Subject(s)
Immunoglobulin Fragments/genetics , Luminescent Proteins/genetics , Amino Acid Sequence , Animals , Antibody Specificity , Base Sequence , Burkholderia/immunology , DNA Primers/genetics , DNA, Recombinant/genetics , Flow Cytometry , Gene Expression , Green Fluorescent Proteins , Immunoglobulin Fragments/biosynthesis , Immunoglobulin Fragments/immunology , Lipopolysaccharides/immunology , Luminescent Proteins/biosynthesis , Luminescent Proteins/immunology , Microscopy, Fluorescence , Molecular Sequence Data , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunologyABSTRACT
The vector pSKAP/S was constructed to enable overexpression of single-chain variable fragment antibody (scFv)-alkaline phosphatase fusion proteins. In pSKAP/S, the scFv were genetically fused to the mutated Escherichia coli PhoA/S gene that encodes an alkaline phosphatase with increased specific activity. The restriction sites incorporated into pSKAP/S allowed the scFv genes to be easily transferred from pUC119-derived phagemid vectors that are used frequently in phage display antibody library technology. Strong transcriptional control of expression was achieved using the tetracycline promoter, and induction of different individual clones with anhydrotetracycline resulted in secretion of most of the scFv-alkaline phosphatase fusion proteins into the culture medium. Although some of the clones secreted fusion proteins that were retained in the periplasm, these proteins could be isolated with a simple extraction procedure. Increased amounts of a scFv-alkaline phosphatase fusion protein were obtained when expressed in the pSKAP/S vector compared with expression in a vector incorporating the lac promoter. Testing for binding of the scFv-alkaline phosphatase fusion proteins to antigen was possible in an ELISA without the need for additional enzyme-conjugated antibodies. The pSKAP/S vector was successfully used to obtain scFv fragments from a preparation of phage-antibody clones after subcloning and expression of individual clones as scFv-alkaline phosphatase fusions, whereas fewer clones (and clones with different properties) were obtained from the same phage-antibody preparations when expressed as soluble scFv fragments. Therefore, the pSKAP/S vector was shown to be useful in extending the range of scFv obtained from phage display libraries.
Subject(s)
Alkaline Phosphatase/genetics , Genetic Vectors , Immunoglobulin Variable Region/genetics , Alkaline Phosphatase/biosynthesis , Alkaline Phosphatase/isolation & purification , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA, Recombinant/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Gene Expression , Genes, Bacterial , Genes, Immunoglobulin , Immunoglobulin Variable Region/biosynthesis , Immunoglobulin Variable Region/isolation & purification , Molecular Sequence Data , Mutation , Protein Folding , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purificationABSTRACT
ABSTRACT Recombinant single-chain antibodies (scFvs) against the lipopolysaccharide of Ralstonia solanacearum (biovar 2, race 3) were successfully selected by phage display from a large combinatorial antibody library. Characterization with regard to cross-reaction and use in routine immunoassays showed that the selected antibodies had improved characteristics when compared with the polyclonal antiserum that is currently used for brown rot diagnosis of potato in the Netherlands. The isolated monoclonal scFvs reacted in both enzyme-linked immunosorbent assay (ELISA) and immunofluorescence cell staining with all race 3 strains tested, but with only some strains belonging to other races. Furthermore, only a few cross-reactions with saprophytic bacteria, which also cross-reacted with polyclonal antisera, were observed. Using ELISA, one of the recombinant antibodies detected as few as 5 x 10(3) bacteria in potato tuber extracts. Therefore, this antibody is potentially useful for detection of R. solanacearum race 3.
