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  • 1
    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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  • 2
    Electronic Resource
    Electronic Resource
    cDNA and protein sequence of a major pea seed albumin (PA 2 : Mr≈26 000) (1987)
    Springer
    Plant molecular biology 8 (1987), S. 37-45 
    Details
    ISSN: 1573-5028
    Keywords: amino acid sequence ; pea seed albumin ; internally repeated sequences
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Pea albumin 2 (PA2:Mr≈26000) is a major component of the albumin fraction derived from aqueous salt extracts of pea seed. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and chromatography on DEAE-Sephacel resolve PA2 into two closely related components (PA2a and PA2b). A cDNA clone coding for one of these components has been sequenced and the deduced amino acid sequence compared with partial, chemically-determined sequences for cyanogen bromide peptides from both PA2 components. Complete amino acid sequences were obtained for the C-terminal peptides. The PA2 molecule of 230 amino acids contains four imperfect repeat sequences each of approximately 57 amino acids in length. The combined sequence data, together with a comparison of PA2-related polypeptides produced in vitro and in vivo, indicate that PA2 is synthesized without a signal sequence and does not undergo significant post-translational modification. Although both forms of PA2 contain Asn-X-Thr consensus sequences, neither form is glycosylated. Accumulation of PA2 contributes approximately 11% of the sulfur-amino acids in pea seeds (cysteine plus methionine equals 2.6 residues percent). Suppression of levels of PA2 polypeptides and their mRNAs in developing seeds of sulfur-deficient plants is less marked than that for legumin, in spite of the lower content of sulfur-amino acids in legumin.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00016432
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  • 3
    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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