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

• Published in: FEBS letters Vol. 441; no. 3; pp. 458 - 462
• Main Authors: Martsev, Sergey P., Kravchuk, Zinaida I., Chumanevich, Alexander A., Vlasov, Alexander P., Dubnovitsky, Anatoly P., Bespalov, Ivan A., Arosio, Paolo, Deyev, Sergey M.
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 28-12-1998
• Subjects: Amino Acid Sequence; Antibodies - genetics; Antibodies - metabolism; Antibody folding and stability; Antigen-binding domain; Base Sequence; Cloning, Molecular; DNA Primers; Escherichia coli - genetics; Ferritins - immunology; Ferritins - metabolism; Human ferritin; Humans; Molecular Sequence Data; Plasmids; Protein Binding; Protein Folding; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Single-chain antibody; Single-chain antibody; Antibody folding and stability; Antigen-binding domain; Human ferritin
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(98)01601-9

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, (Gly
4Ser)
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=5.1×10
8 M
−1 in RIA and 1.8×10
8 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.