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  • dimerization  (2)
  • anti-idiotype  (1)
  • 1
    Electronic Resource
    Electronic Resource
    Solution properties ofEscherichia coli-expressed VH domain of anti-neuraminidase antibody NC41 (1995)
    Springer
    The protein journal 14 (1995), S. 167-178 
    Details
    ISSN: 1573-4943
    Keywords: Antibody ; VH domain ; dimerization ; detergent stabilization of monomer ; NMR analysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract The VH domain of anti-influenza neuraminidase antibody NC41, with and without a C-terminal hydrophilic marker peptide (FLAGTM), has been expressed in high yield (15–27 mg/L) inEscherichia coli. Both forms were secreted into the periplasm where they formed insoluble aggregates which were solubilized quantitatively with 2 M guanidine hydrochloride and purified to homogeneity by ion-exchange chromatography. The VH-FLAG was composed of three isoforms (pI values of ∼4.6, 4.9, and 5.3) and the VH molecule was composed of two isoforms with pI values of 5.1 and 6.7; the difference between the VH isoforms was shown to be due to cyclization of the N-terminal glutamine residue in the pI 5.1 isoform. At 20°C and concentrations of 5–10mg/ml the VH domain dimerized in solution and then partly precipitated, resulting in the broadening of resonances in its1H NMR spectrum. Reagents such as CHAPS,n-octylglucoside, and ethylene glycol, which presumably mask the exposed hydrophobic interface of the VH molecule, prevented dimerization of the VH and permitted good-quality NMR spectra on isotope-labeled protein to be obtained.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01980329
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  • 2
    Electronic Resource
    Electronic Resource
    Utilization of the Streptoalloteichus hindustanus Resistance Determinant ShBle as a Protein Framework: Effect of Mutation upon ShBle Dimerization and Interaction of C-Terminal Displayed Peptide Epitopes (1999)
    Springer
    The protein journal 18 (1999), S. 813-821 
    Details
    ISSN: 1573-4943
    Keywords: ShBle ; framework ; dimerization ; FLAG epitope tag
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract We have selected the Streptoalloteichus hindustanus bleomycin-resistance protein ShBle, a 28-kDa homodimer, as a scaffold for the display of bioactive peptides and other peptide epitopes. To create a monomeric scaffold, we investigated the effect of mutating residue proline 9 to glycine. This residue plays a critical role in ShBle dimerization by affecting the position of the eight N-terminal residues which secure the interaction between the monomeric subunits. We demonstrate that this mutation weakens the dimerization interaction, resulting in establishment of a stable equilibrium between monomeric and dimeric ShBle species in solution. Circular dichroism and SDS–PAGE data indicate that the Pro9Gly mutation does not disrupt the structure of the molecule. Production of a fully monomeric form of ShBle required complete removal of the eight-residue N-terminal peptide, and the interaction across the now solvent-exposed hydrophobic interface of the ShBle monomer was insufficient to drive dimerization. To demonstrate efficient display of epitope tags on the ShBle protein, we displayed dual-octapeptide FLAG tags at the protein C-terminus. These additions did not interfere with protein folding or activity. The resulting ShBle scaffold was used to compare the efficiency of two commercial FLAG-specific antibodies by biosensor.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1020618910455
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  • 3
    Electronic Resource
    Electronic Resource
    Single-Chain Fv of Anti-idiotype 11-1G10 Antibody Interacts with Antibody NC41 Single-Chain Fv with a Higher Affinity than the Affinity for the Interaction of the Parent Fab Fragments (1998)
    Springer
    The protein journal 17 (1998), S. 245-254 
    Details
    ISSN: 1573-4943
    Keywords: Antibody ; anti-idiotype ; single-chain Fvs ; solution properties ; complexes with antigens ; binding affinities
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract A single-chain Fv (scFv) fragment of anti-idiotype antibody 11-1G10, which recognizes an idiotope of anti-neuraminidase antibody NC41, was constructed by joining VH and VL domains with a (Gly4Ser)3 linker, with a pelB leader sequence, and two C-terminal FLAG™ tag sequences, and expressed in E. coli (10 mg/L). The 11-1G10 scFv was isolated by affinity chromatography on an anti-FLAG M2 antibody column as a 2:1 mixture of monomer and dimer forms which were separated by Superdex 75 chromatography; monomer (at 100 μg/ml) was stable for 7 days at 21°C and 30 days at 4°C, whereas the dimer slowly dissociated to monomer to yield a 2:1 monomer–dimer equilibrium mixture after 30 days at 4°C. The dimer was bivalent, with each combining site binding an NC41 Fab to yield a stable complex of M r ≍ 156,000. Binding affinities, determined in solution using a BIAcore™ biosensor, showed that the affinity for the interaction of 11-1G10 scFv monomer with NC41 scFv monomer was five- to six-fold higher than the interaction of the parent Fab pair. This is the first example of an scFv derived from a monoclonal antibody with a higher affinity than its parent Fab.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1022584702108
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