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

The pET(scF11) plasmid mas constructed comprising the gene of a single-chai n antibody against human ferritin, This plasmid encodes the leader peptide pelB followed by the heavy chain variable V-H domain, (Gly(4)Ser)(3) linker peptide, and light chain variable V-L domain. The correctly processed scF1 1 antibody was expressed in Escherichia coli as an insoluble protein withou t the leader peptide. Purified soluble scF11 mas obtained after solubilizat ion in 6 M GdnHCl followed by a sequential dialysis against decreasing urea concentrations and ion-exchange chromatography. ScF11 demonstrated only a similar to 8-fold decrease in the affinity (K-a=5.1x10(8) M-1 in RIA and 1. 8x10(8) M-1 in ELISA) vs. the parent IgG2a/kappa monoclonal antibody F11, T he emission maximum of intrinsic fluorescence strongly suggests a compact c onformation with tryptophanyl fluorophores buried in the protein interior, consistent with the functionality of the protein, However, scF11 demonstrat ed (i) the lack of denaturant-induced fluorescence 'dequenching' effect cha racteristic of the completely folded parent antibody, and (ii) prominent bi nding, under physiological conditions, of a hydrophobic probe 8-anilino-1-n aphthalenesulfonate (ANS) recognizing partially structured states of a prot ein. These findings are indicative of an incomplete tertiary fold that give s ANS access to the protein hydrophobic core. This work provides the first indication that the functional single-chain antibody scF11 displays some pr operties of a partially structured state and therefore may possess incomple te folding. (C) 1998 Federation of European Biochemical Societies.