AFFINITY AND FOLDING PROPERTIES BOTH INFLUENCE THE SELECTION OF ANTIBODIES WITH THE SELECTIVELY INFECTIVE PHAGE (SIP) METHODOLOGY

We investigated which molecules are selected from a model library by t he selectively infective phage (SIP) methodology. As a model system, w e used the fluorescein binding single-chain Fv fragment FITC-E2, and f rom a 3D-model, we identified 11 residues potentially involved in hapt en binding and mutated them individually to alanines. The binding cons tant of each mutant was determined by fluorescence titration, and each mutant was tested individually as web as in competitive STP experimen ts for infectivity. After three rounds of SIP, only molecules with K-D values within a factor of 2 of the tightest binder remain, and among those, a mutant no longer carrying an unnecessary exposed tryptophan r esidue is preferentially selected. SIP is shown to select for the best overall properties of the displayed molecules, including folding beha vior, stability and affinity. (C) 1997 Federation of European Biochemi cal Societies.