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  • Biochemistry and Biotechnology  (2)
  • antibody  (2)
  • phosphorylation  (1)
  • Exotoxin
  • Chemotherapy
  • Wiley-Blackwell  (3)
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  • 1
    ISSN: 0887-3585
    Keywords: antibody ; antitumor ; single chain Fv ; variable domains ; X-ray crystallography ; protein structure ; protein stability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A recombinant Fv construct of the B1 monoclonal antibody that recognizes the LewisY-related carbohydrate epitope on human carcinoma cells has been prepared. The Fv is composed of the polypeptide chains of the VH and VL domains expressed independently and isolated as inclusion bodies. The Fv is prepared by combining and refolding equimolar amounts of guanidine chloride solubilized inclusion bodies. The Fv is stabilized by an engineered interchain disulfide bridge between residues VL100 and VH44. This construct has a similar binding affinity as that of the single-chain construct (Benhar and Pastan, Clin. Cancer Res. 1:1023-1029, 1995). The B1 disulfide-stabilized Fv (B1dsFv) crystallizes in space group P6122 with the unit cell parameters a = b = 80.1 Å, and c = 138.1 Å. The crystal structure of the B1dsFv has been determined at 2.1-Å resolution using the molecular replacement technique. The final structure has a crystallographic R-value of 0.187 with a root mean square deviation in bond distance of 0.014 Å and in bond angle of 2.74°. Comparisons of the B1dsFv structure with known structures of Fv regions of other immunoglobulin fragments shows closely related secondary and tertiary structures. The antigen combining site of B1dsFv is a deep depression 10-Å wide and 17-Å long with the walls of the depression composed of residues, many of which are tyrosines, from complementarity determining regions L1, L3, H1, H2, and H3. Model building studies indicate that the LewisY tetrasaccharide, Fuc-Gal-Nag-Fuc, can be accommodated in the antigen combining site in a manner consistent with the epitope predicted in earlier biochemical studies (Pastan, Lovelace, Gallo, Rutherford, Magnani, and Willingham, Cancer Res. 51:3781-3787, 1991). Thus, the engineered disulfide bridge appears to cause little, if any, distortion in the Fv structure, making it an effective substitute for the B1 Fab. Proteins 31:128-138, 1998. Published 1998 Wiley-Liss, Inc.This article is a US Government work and, as such, is in the public domain in the United States of America.
    Additional Material: 10 Ill.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 0730-2312
    Keywords: insulin receptor ; tyrosine kinase ; pp60src ; phosphorylation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Both the insulin receptor and the gene product of the Rous sarcoma virus, pp60src, are protein kinases which phosphorylate themselves and other proteins on tyrosinc residues. Addition of the solubilized insulin receptor to purified pp60src increased the phosphorylation of the β-subunit of the insulin receptor. Phosphorylation of the insulin receptor by pp60src occurred both in the absence and presence of insulin but did not alter the insulin dose response for autophosphorylation of the receptor. Increasing concentrations of pp60src increased the phosphorylation of the receptor and at high concentrations equaled the maximal effect produced by insulin. Our observations suggest a possible mechanism by which the metabolically regulated insulin receptor tyrosine kinase could be altered by other tyrosine kinases such as that associated with pp60src. Further studies will be required to determine if the insulin receptor is phosphorylated by pp60src in Rous sarcoma virus-infected cells.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 0887-3585
    Keywords: immunoglobulin ; antibody ; mAb B3 ; protein engineering ; disulfide bond ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The Fv fragments are the smallest units of antibodies that retain the specific antigen binding characteristics of the whole molecule and are being used for the diagnosis and therapy of human diseases. These are noncovalently associated heterodimers of the heavy (V H) and the light (VL) chain variable domains, which, without modification, tend to dissociate, unfold, and/or nonspecific ally aggregate. The fragment is usually stabilized by producing it as a single chain recombinant molecule in which the two chains are linked by means of a short polypeptide linker. An alternative strategy is to connect the two chains by means of an interchain disulfide bond. We used molecular graphics and other modeling tools to identify two possible interchain disulfide bond sites in the framework region of the Fv fragment of the monoclonal mouse antibody (mAb) B3. The mAb B3 binds to many human cancer cells and is being used in the development of a new anticancer agent. The two sites identified are VH44-VL105 and VH111-VL48. (VH44-VL100 and VH105-VL43 in the numbering scheme of Kabat et al., “Sequence of Proteins of Immunological Interest,” U.S. DHHS, NIH publication No. 91-3242, 1991.) This design was recently tested using the chimeric protein composed of a truncated form of Pseudomonas exotoxin and the Fv fragment of mAb B3 with the engineered disulfide bond at VH44-VL105 (Brinkmann et al., Proc. Natl. Acad. Sci. U.S.A. 90:7538, 1993). The chimeric toxin was found to be just as active as the corresponding single chain counterpart and considerably more stable. Because these disulfide bond sites are in the framework region, they can be located from sequence alignment alone. We expect that the disulfide bond at these sites will stabilize the Fv fragment of most antibodies and the antigen-specific portion of the T-cell receptors, which are homologous. © 1994 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
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