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At least 595 records · Page 33Linked to original sources
PubMed1994

Spontaneous assembly of bivalent single chain antibody fragments in Escherichia coli.

The ability of immunoglobulin Fab and single chain (ScFv) fragments to penetrate effectively into tissue from the vascular system has made these molecules excellent candidates as drug delivery systems and imaging tools. This study investigates the use of single chain antibody fragment bacterial expression vectors as a possible strategy for the production of these molecules. We have modified the pSW1-VHD1.3-VKD1.3-TAG1 vector [Ward et al. (1989) Nature 341, 544-546] which originally, when expressed in E. coli, produced an Fab fragment. In an effort to improve the affinity of the parent vector product a novel single chain antibody construct which encodes a protein with anti-P. aeruginosa activity was generated using a 14 amino acid linker [Chaudhary et al. (1990) Proc. natn. Acad. Sci. U.S.A. 87, 1066-1070]. In addition to the heavy and light chain variable domain genes, our construct also contained the light chain kappa constant domain gene to aid purification of the fragments. To underline this difference from the conventional ScFv fragment we have described this protein as a ScAb. The ScAb generated had an antigen binding capacity similar to the parent anti-P. aeruginosa antibody but was superior to the recombinant anti-P. aeruginosa Fab fragment. On HPLC and non-denaturing gel electrophoresis analysis, the ScAb was found to exist in multimeric forms while the Fab fragment existed only as a single unit. Dimeric ScAb had a similar antigen binding profile to the parent antibody.

Antibodies↗
PubMed1994

Recombinant single-chain Fv fragments carrying C-terminal cysteine residues: production of bivalent and biotinylated miniantibodies.

A murine antibody single-chain Fv (scFv) fragment carrying five C-terminal histidine residues preceded by a cysteine residue and a marker peptide was expressed in Escherichia coli. Its variable heavy (VH) and light (VL) domains are derived from the mouse monoclonal antibody mAb215, which is specific for the largest subunit of RNA polymerase II of Drosophila melanogaster. ScFv' monomers, covalently linked (scFv')2 and non-covalent dimers, as well as aggregated antibody fragments, were isolated from an E. coli cell paste by immobilized metal affinity chromatography in 6 M urea followed by a renaturation procedure that does not use any sulfhydryl agents. In a final step, the components were separated by size exclusion chromatography. All the recombinant antibody fractions demonstrated high antigen-binding activity and specificity as shown by ELISA and Western blot analysis. Affinity measurements carried out by competitive immunoassays showed that covalently linked (scFv')2 have binding constants quite close to those of the parental monoclonal antibodies and four-fold higher than scFv' monomers. ScFv derivatives, specifically biotinylated through the free sulfhydryl group, recognize the corresponding antigen in ELISA and Western blot analysis, thus demonstrating the possibility of using chemically modified scFv antibodies for immunodetection.

Amino Acid Sequence↗
PubMed1985

Studies of bivalent copper ion binding to poly C.

Ultraviolet differential spectra of single-stranded poly C, taken in the presence of Cu2+ ions, are studied at various ionic strengths and temperatures. Coordinational and conformational components of these spectra are obtained. The Cu2+ ion coordination site on the polynucleotide bases is found to be N(3) and possibly O(2). The direction of the poly C absorption band shift due to ion binding and conformational transitions is established. At low ionic strengths of the solution Cu2+ ions cause the helical parts of poly C to melt. At high ones the formation of double-stranded parts was observed in addition to the above effect. The calculated concentration dependences of ion-poly C bases association constants show that binding is cooperative at any ionic strength.

Copper↗
PubMed1987

Studies of formation of bivalent copper complexes with native and denatured DNA.

The formation of Cu2+ complexes with native and denatured DNA is studied by the methods of differential UV spectroscopy, CD spectroscopy, and viscometry. On ion binding to the bases of native DNA the latter transforms into a new conformation. This transition is accompanied with a sharp increase in UV absorption and a decrease in the intrinsic viscosity though the high degree of helicity persists. Possible sites of Cu2+ ion binding on DNA of various conformations are found along with corresponding constants of complex formation.

Circular Dichroism↗
PubMed1996

Protein interactions with bivalent tin. 1. Hydrolysis and complexation of tin(II) ion with glycine.

The complexation between tin(II) ion and glycine was studied in 0.15 mol/dm3 NaCl medium at 310 K using potentiometric glass electrode titrations. In the pH range 1.1-4.5 and concentration range of the tin(II) between 0.2 and 5.0 mmol/dm3, with variable glycine-to-tin molar ratio up to 10:1, the experimental data were explained by the formation of the following complexes and their overall stability constants: log(beta +/- sigma): Sn(HGly)+, (12.78 +/- 0.08); Sn(Gly)+, (10.02 +/- 0.07); Sn(OH)Gly, (7.34 +/- 0.03), as well as the pure hydrolytic complex Sn4(OH)2+(6), whose stability constant was determined in separate experiments and found to be -4.30 +/- 0.08, under the same experimental conditions as for complexation study. The precipitate formed in tin(II)-glycine system at pH ca. 5.0 was characterized by chemical and TG analysis, I. R. spectra, X-ray powder diffraction, and electron scanning microscopy measurements. It has been shown that the precipitate has the composition Sn(OH)Gly and crystallizes in a tetragonal system with unit cell dimensions a = b = 1.584 nm, c = 0.597 nm. The mechanism of the complex formation in solution is discussed.

Cations, Divalent↗
PubMed1996

Interaction of bivalent copper, nickel, manganese ions with native DNA and its monomers.

UV differential spectroscopy is applied to study the interaction of Cu2+, Ni2+, Mn2+ ions with deoxyribonucleotides of canonic bases (dGMP, dAMP, dCMP, dTMP) and native DNA. Heteroatoms of the bases, coordinating ions, and binding constants which characterize the formation of metal complexes are found. The affinity of the ions is lower for the deoxyribonucleotide bases than for the ribonucleotide ones. This indicates that 02' of ribose participates in the stabilization of the metal complex even under conditions close to the neutral one (pH 6). Unlike the Cu2+ ions, Ni2+ and Mn2+ ions do not interact with N3C both in monomers and polymers. This seems to be the main factor explaining why copper makes DNA transform into a structure with a quasi-Hoogsteen pairing of GC pairs. No transformations of this kind of helix-coil transitions are caused by manganese and nickel up to concentrations 4 X 10(-2) M.

Copper↗
PubMed1989

A comparison of the antioxidant requirements of proteins with those of synthetic polymers suggests an antioxidant function for clusters of aromatic and bivalent sulphur-containing amino acid residues.

Many proteins which function in extracellular environments potentially rich in oxygen-derived free radicals contain clustered tyrosine and cysteine residues which, by analogy with the chemistry of antioxidants used with synthetic polymers, may provide an appreciable antioxidant and redox stabilization activity. Such proteins may function as antioxidants, and as ligand binding sites for free radicals and other active molecules employed in normal biochemical processes.

Antioxidants↗
PubMed2004

A bivalent single-chain Fv fragment against CD47 induces apoptosis for leukemic cells.

We constructed a single-chain antibody fragment (scFv) of murine monoclonal antibody, MABL, which specifically bound to human CD47 (hCD47) and induced apoptosis of the leukemic cells. The scFv of MABL antibody with a 15-residue linker (MABL scFv-15) formed both dimer (Mr 50 kDa) and monomer (Mr 25 kDa). Both MABL scFv-15 dimer and monomer had binding activity for hCD47. MABL scFv-15 dimer strongly induced apoptosis of hCD47-introduced mouse leukemic cells in vitro and exhibited anti-tumor effect in a myeloma transplanted mice model. However, MABL scFv-15 monomer scarcely exhibited these activities. These results strongly demonstrate that the ligation of CD47 antigen by two antigen-binding sites of MABL dimer is needed for inducing apoptosis. The parent MABL antibody caused hemagglutination due to the CD47 expressed on erythrocytes. Interestingly, MABL scFv-15 dimer did not cause hemagglutination. This apoptosis-inducing dimer appears to be a lead candidate for novel leukemic therapy.

Animals↗