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

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, (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.