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

FEBS Letters

Deyev, Sergey M.²  Chumanevich, Alexander A.¹  Dubnovitsky, Anatoly P.¹  Martsev, Sergey P.¹  Vlasov, Alexander P.¹  Bespalov, Ivan A.¹  Arosio, Paolo³  Kravchuk, Zinaida I.¹ 

[1] Institute of Bio-Organic Chemistry, National Academy of Sciences of Belarus, Zhodinskaya 5/2, Minsk 220141, Belarus;Engelhardt Institute of Molecular Biology, and Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia;DIBIT, Scientific Institute H. San Raffaele, Milano, Italy

关键词: Single-chain antibody;    Human ferritin;    Antibody folding and stability;    Antigen-binding domain;   

DOI  :  10.1016/S0014-5793(98)01601-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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=5.1×10⁸ M⁻¹ in RIA and 1.8×10⁸ 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.

【 授权许可】

Unknown