Reproducing the natural evolution of protein structural features with the selectively infective phage (SIP) technology. the kink in the first strand of antibody kappa domains

Abstract

The β-sandwich structure of immunoglobulin variable domains is characterized by a typical kink in the first strand, which allows the first part of the strand to hydrogen bond to the outer β-sheet (away from the VH-VL interface) and the second part to the inner β-sheet. This kink differs in length and sequence between the Vκ, Vλ and VH domains and yet is involved in several almost perfectly conserved interactions with framework residues. We have used the selectively infective phage (SIP) system to select the optimal kink region from several defined libraries, using an anti-hemagglutinin single-chain Fv (scFv) fragment as a model system. Both for the kink with the Vκ domain length and that with the Vλ length, a sequence distribution was selected that coincides remarkably well with the sequence distribution of natural antibodies. The selected scFv fragments were purified and characterized, and thermodynamic stability was found to be the prime factor responsible for selection. These data show that the SIP technology can be used for optimizing protein structural features by evolutionary approaches.