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Recombinant fragments of the variable region of antibodies are useful in many experimental and clinical applications. However, it can be difficult to obtain these materials in soluble form after their expression in bacteria. Here, we report an efficient procedure for preparing several variable-domain fragments (Fv), single-chain Fv (scFv), and a diabody (the smallest functional bispecific antibody) of anti-carcinoembryonic antigen (CEA) antibody by overexpression in Escherichia coli in inclusion bodies, using a refolding system to obtain renatured proteins. Two types of refolded Fv were prepared: (i) Heavy and light chains of the immunoglobulin variable regions (VH and VL, respectively) were coexpressed with a dicistronic expression vector (designated Fvco); (ii) VH and VL were expressed separately, mixed stoichiometrically, and refolded (designated Fvmix). All samples refolded with high efficiency; Fvco, Fvmix, seFv, and the bispecific diabody bound to several CEA-positive cell lines, exactly as did soluble Fv fragments secreted by E. coli (Fvsol) and the parent IgG. The refolded fragments inhibited binding of the parent IgG to CEA-positive cell lines, indicating that their epitope is identical to that of IgG. The bispecific diabody, which combined variable-region fragments of anti-CEA antibody with variable-region fragments of anti-CD3 antibody, was also prepared using the refolding system. This refolded diabody could bind to lymphokine-activated killer cells. In addition, its cytotoxicity toward human bile duct carcinoma TFK-1 and other several other CEA-positive cell lines was concentration-dependent. Taken together, our results suggest that a refolding procedure can be used to prepare various functional antibody fragments (Fv, scFv, and diabody). [ABSTRACT FROM AUTHOR]

We report a strategy for generating efficient signal transduction with unnatural heterol-ogous receptor combinations. As previously described [Ueda, H., Kawahara, M. et aL (2000) J. Immunol. Methods 241, 159-170], chimeric receptors composed of the VR/VL domains of anti-hen egg lysozyme antibody HyHEL-10 and N-tenninally truncated erythropoietin receptor (EpoR) can be activated by lysozyme. When the cytoplasmic domains of these receptors were substituted with one derived from gpl30, IL-3 dependent Ba/F3 cells expressing both VH-gpl30 and VL-gpl30 grew dose-dependently when given lysozyme without IL-3. However, cells expressing the heterologous pair of VH-gpl30 and VL-EpoR also showed more efficient and stricter lysozyme-dependent proliferation in the absence of IL-3, indicating this combination is as an efficient and strict signal transducer as wild-type EpoR. The immunoprecipitation data indicated the existence of a preformed VH-gpl30 and VL-EpoR heterodimer in the absence of lysozyme, suggesting the crucial role of a receptor conformational change in signal triggering as well as wild-type EpoR and gpl30. Phosphorylation of JAK2, STAT3, and STAT5 was observed upon the addition of lysozyme, suggesting the activation of both EpoR- and gpl30-derived signals. [ABSTRACT FROM AUTHOR]

We identified a gene encoding a catalase from the anaerobic bacteria Desulfovibrio vulgaris (Miyazaki F), and the expression of its gene in Escherichia coli. The 3.3-kbp DNA fragment isolated from D. vulgaris (Miyazaki F) by double digestion with EcoRI and SalI was found to produce a protein that binds protoheme IX as a prosthetic group in E. coli. This DNA fragment contained a putative open reading frame (Kat) and one part of another open reading frame (ORF-1). The amino acid sequence of the amino terminus of the protein purified from the transformed cells was consistent with that deduced from the nucleotide sequence of Kat in the cloned fragment of D. vulgaris (Miyazaki F) DNA, which may include promoter and regulatory sequences. The nucleotide sequence of Kat indicates that the protein is composed of 479 amino acids per monomer. The recombinant catalase was found to be active in the decomposition of hydrogen peroxide, as are other catalases from aerobic organisms, but its Km value was much greater. The hydrogen peroxide stress against D. vulgaris (Miyazaki F) induced the activity for the decomposition of hydrogen peroxide somewhat, so the catalase gene may not work effectively in vivo. [ABSTRACT FROM AUTHOR]

We have established a method for selecting binding phages from a phage immunoglobu-lin heavy chain variable region (VH) library by panning with nitrocellulose membranes (membrane panning). To evaluate the concentrating ability of membrane panning for binding phages, a phage VH library containing clones that bind to hen egg white lyso-zyme (HEL) was used for panning against HEL. The efficiency of our method was as high as that of panning with magnetic beads. In addition, we performed membrane panning against target proteins and isolated the binding phages. The human VH genes of these phages were cloned and expressed as VH-bacterial alkaline phosphatase (PhoA) conjugates (VH-PhoA) in Escherichia coli. The dose-dependent binding of VH-PhoA to target proteins was confirmed by dot blotting. When applied to disease-associated antibodies, these methods will likely benefit clinical research. In addition, these techniques may be applicable to systematic analysis in proteome studies. [ABSTRACT FROM AUTHOR]

A bacterial expression system for the variable region fragments (Fvs) of the anti-MUC1 tumor antigen antibody MUSE11 has been constructed. The Fv fragment showed binding specificity toward TFK-1 cells, with slightly reduced affinity compared to its parent IgG. The single-chain Fv fragment was arranged in two orders, VH-linker-VL and VL-linker-VH. However, linking the regions with a flexible peptide linker (GGGGS)3 or with a shorter linker (GGGGS) led to a dramatic decrease in the biological activity toward the target antigen in both arrangements, suggesting that the MUSE11 antibody loses its activity when the domains are linked with polypeptide linkers. These results indicate that the variable region domains of the anti-MUC1 antibody MUSE11 have specificity only in the Fv form, and that linking the domains strongly reduces the association with its target antigen. Gel filtration analysis indicates that the scFv has a dimeric structure, suggesting that the inactivation of MUSE11 scFv is due to unfavorable intermolecular associations of the scFv chains. To our knowledge, this is the first report of a significant reduction in affinity caused by linking the variable domains in both arrangements, i.e., VH-VL and VL-VH. [ABSTRACT FROM AUTHOR]

Functional structures of hairpin ribozymes have been investigated by constructing various chemically modified molecules. Domain-exchange and linker insertion experiments were performed to find active conformations of the RNA enzyme showing cleavage activity. Our experiments and other evidence suggest that the active structure has a bent conformation, and that domain-interactions are essential for the cleavage activity. [ABSTRACT FROM AUTHOR]

Single-chain antibodies (scFv), which can be produced in Escherichia coli cells, have been shown in numerous cases to be active in antigen binding. In the case of the two anti-lyso-zyme single-chain antibodies, scFvLH and scFvHL which have the reversed arrangement of the variable domains of the heavy and light chains of the corresponding monoclonal antibodies, the expression level differs greatly when they are produced in Escherichia coli [Tsumoto et al. (1995) Biochem. Biophys. Res. Commun. 201, 546–551]. Although the expression level of scFvLH is high in vivo, the single chain antibody with the reversed orientation (scFvHL) was synthesized in a very low yield and no active product could be obtained. We report here the synthesis of these two anti-lysozyme single-chain antibodies in high yields and with high biological activities in a cell-free E. coli expression system in the presence of reduced and oxidized glutathione, protein disulfide isomerase (PDI), and chaperones. In immunological blotting assays, the synthesized scFvs with both arrangements exhibit specific binding activity to the corresponding antigens, hen egg-white lysozyme, and in an activity assay both inhibited the action of lysozyme. scFvLH is synthesized mainly as a product with the expected molecular weight, whereas scFvHL is produced with additional shorter fragments, suggesting that the low yield isolation through the expression in vivo is due to mistranslation or ribonucleolytic cleavage of the transcript. In the cell-free expression of scFv a certain amount of the product is precipitated but in the presence of chaperones the amount of soluble protein increased from 25 to 90% (PDI and chaperones). The overall expression level and the specific biological activity, however, were hardly influenced. The system reported here can provide significant amounts of various scFv fragments regardless of the order of variable regions, including those which are hardly expressed in vivo. [ABSTRACT FROM PUBLISHER]

The gene encoding a flavodoxin of Desulfovibrio vulgaris (Miyazaki F) was cloned, and overexpressed in Escherichia coli. A 1.6-kbp DNA fragment, isolated from D. vulgaris (Miyazaki F) by double digestion with SalI and EcoRI, contained the flavodoxin gene and its regulatory region. An expression system for the flavodoxin gene under control of the T7 promoter was constructed in E. coli. The purified protein was soluble and exhibited a characteristic visible absorption spectrum. HPLC analysis of the recombinant flavodoxin revealed the presence of an identical FMN to that found in the native D. vulgaris flavodoxin, and its dissociation constant with FMN was determined to be 0.38 nM. In vitro H2 reduction analysis indicated that the recombinant flavodoxin is active, and its redox potential was determined to be E1 = −434 and E2 = −151 mV using methyl viologen and 2-hydroxy-1,4-naphthoquinone, respectively. Its redox behavior was also examined with the recombinant flavodoxin adsorbed onto a graphite electrode. The mutant, A16E, was also produced, which revealed the feature of a conserved Glu residue at the surface of the molecule. [ABSTRACT FROM AUTHOR]

Biomolecules which recognize inorganic materials and metal surfaces gain much attention for creating new type of nanomaterials and sensors. 4F2, a camelid VHH antibody, recognizes ZnO surface and has been applied for sensor applications. 4F2 was constructed sequential complementarity determining region (CDR) replacement on the parental VHH antibody, termed the Construction of Antibody by Integrating Grafting and Evolution Technology; CAnIGET procedure. Here, we evaluate the influence of CDR replacements during 4F2 generation using calorimetric technique. We found that the initial peptide grafting at CDR1 results in the stability reduction and subsequent CDR3 randomize and selection restore the stability during the construction of 4F2. Further examination using anti-gold VHH, AuE32, revealed that the final CDR3 randomize and selection step has little effect in stability while the initial CDR1 grafting reduces the stability as same as the case for 4F2. Our results showing here provide the detailed view of the stability alteration during the CAnIGET procedure.