Antiferritin single-chain antibody: A functional protein with incomplete folding?

Sergey P. Martsev, Zinaida I. Kravchuk, Alexander A. Chumanevich, Alexander P. Vlasov, Anatoly P. Dubnovitsky, Ivan A. Bespalov, Paolo Arosio, Sergey M. Deyev

    Research output: Contribution to journalArticle

    22 Citations (Scopus)

    Abstract

    The pET(scF11) plasmid was constructed comprising the gene of a single-chain antibody against human ferritin. This plasmid encodes the leader peptide pelB followed by the heavy chain variable V(H) domain, (Gly4Ser)3 linker peptide, and light chain variable V(L) domain. The correctly processed scF11 antibody was expressed in Escherichia coli as an insoluble protein without the leader peptide. Purified soluble scF11 was obtained after solubilization in 6 M GdnHCl followed by a sequential dialysis against decreasing urea concentrations and ion-exchange chromatography. ScF11 demonstrated only a ~8-fold decrease in the affinity (K(a)=5.1x108 M-1 in RIA and 1.8x108 M-1 in ELISA) vs. the parent IgG2a/κ monoclonal antibody F11. The emission maximum of intrinsic fluorescence strongly suggests a compact conformation with tryptophanyl fluorophores buried in the protein interior, consistent with the functionality of the protein. However, scF11 demonstrated (i) the lack of denaturant-induced fluorescence 'dequenching' effect characteristic of the completely folded parent antibody, and (ii) prominent binding, under physiological conditions, of a hydrophobic probe 8-anilino-1-naphthalenesulfonate (ANS) recognizing partially structured states of a protein. These findings are indicative of an incomplete tertiary fold that gives ANS access to the protein hydrophobic core. This work provides the first indication that the functional single-chain antibody scF11 displays some properties of a partially structured state and therefore may possess incomplete folding. Copyright (C) 1998 Federation of European Biochemical Societies.

    Original languageEnglish
    Pages (from-to)458-462
    Number of pages5
    JournalFEBS Letters
    Volume441
    Issue number3
    DOIs
    Publication statusPublished - 28 Dec 1998

    Fingerprint

    Single-Chain Antibodies
    Protein Sorting Signals
    Proteins
    Naphthalenesulfonates
    Anilino Naphthalenesulfonates
    Plasmids
    Fluorescence
    Dialysis
    Fluorophores
    Ion Exchange Chromatography
    Ferritins
    Chromatography
    Escherichia coli
    Conformations
    Urea
    Ion exchange
    Genes
    Enzyme-Linked Immunosorbent Assay
    Monoclonal Antibodies
    scF11 antibody

    Keywords

    • Antibody folding and stability
    • Antigen-binding domain
    • Human ferritin
    • Single-chain antibody

    ASJC Scopus subject areas

    • Structural Biology
    • Biophysics
    • Biochemistry
    • Molecular Biology
    • Genetics
    • Cell Biology

    Cite this

    Martsev, S. P., Kravchuk, Z. I., Chumanevich, A. A., Vlasov, A. P., Dubnovitsky, A. P., Bespalov, I. A., ... Deyev, S. M. (1998). Antiferritin single-chain antibody: A functional protein with incomplete folding? FEBS Letters, 441(3), 458-462. https://doi.org/10.1016/S0014-5793(98)01601-9

    Antiferritin single-chain antibody : A functional protein with incomplete folding? / Martsev, Sergey P.; Kravchuk, Zinaida I.; Chumanevich, Alexander A.; Vlasov, Alexander P.; Dubnovitsky, Anatoly P.; Bespalov, Ivan A.; Arosio, Paolo; Deyev, Sergey M.

    In: FEBS Letters, Vol. 441, No. 3, 28.12.1998, p. 458-462.

    Research output: Contribution to journalArticle

    Martsev, SP, Kravchuk, ZI, Chumanevich, AA, Vlasov, AP, Dubnovitsky, AP, Bespalov, IA, Arosio, P & Deyev, SM 1998, 'Antiferritin single-chain antibody: A functional protein with incomplete folding?', FEBS Letters, vol. 441, no. 3, pp. 458-462. https://doi.org/10.1016/S0014-5793(98)01601-9
    Martsev SP, Kravchuk ZI, Chumanevich AA, Vlasov AP, Dubnovitsky AP, Bespalov IA et al. Antiferritin single-chain antibody: A functional protein with incomplete folding? FEBS Letters. 1998 Dec 28;441(3):458-462. https://doi.org/10.1016/S0014-5793(98)01601-9
    Martsev, Sergey P. ; Kravchuk, Zinaida I. ; Chumanevich, Alexander A. ; Vlasov, Alexander P. ; Dubnovitsky, Anatoly P. ; Bespalov, Ivan A. ; Arosio, Paolo ; Deyev, Sergey M. / Antiferritin single-chain antibody : A functional protein with incomplete folding?. In: FEBS Letters. 1998 ; Vol. 441, No. 3. pp. 458-462.
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    AU - Martsev, Sergey P.

    AU - Kravchuk, Zinaida I.

    AU - Chumanevich, Alexander A.

    AU - Vlasov, Alexander P.

    AU - Dubnovitsky, Anatoly P.

    AU - Bespalov, Ivan A.

    AU - Arosio, Paolo

    AU - Deyev, Sergey M.

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    AB - The pET(scF11) plasmid was constructed comprising the gene of a single-chain antibody against human ferritin. This plasmid encodes the leader peptide pelB followed by the heavy chain variable V(H) domain, (Gly4Ser)3 linker peptide, and light chain variable V(L) domain. The correctly processed scF11 antibody was expressed in Escherichia coli as an insoluble protein without the leader peptide. Purified soluble scF11 was obtained after solubilization in 6 M GdnHCl followed by a sequential dialysis against decreasing urea concentrations and ion-exchange chromatography. ScF11 demonstrated only a ~8-fold decrease in the affinity (K(a)=5.1x108 M-1 in RIA and 1.8x108 M-1 in ELISA) vs. the parent IgG2a/κ monoclonal antibody F11. The emission maximum of intrinsic fluorescence strongly suggests a compact conformation with tryptophanyl fluorophores buried in the protein interior, consistent with the functionality of the protein. However, scF11 demonstrated (i) the lack of denaturant-induced fluorescence 'dequenching' effect characteristic of the completely folded parent antibody, and (ii) prominent binding, under physiological conditions, of a hydrophobic probe 8-anilino-1-naphthalenesulfonate (ANS) recognizing partially structured states of a protein. These findings are indicative of an incomplete tertiary fold that gives ANS access to the protein hydrophobic core. This work provides the first indication that the functional single-chain antibody scF11 displays some properties of a partially structured state and therefore may possess incomplete folding. Copyright (C) 1998 Federation of European Biochemical Societies.

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    KW - Antigen-binding domain

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    KW - Single-chain antibody

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