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 the selectively infective phage (SIP) methodology. As a model system, we used the fluorescein binding single-chain Fv fragment FITC-E2, and from a 3D-model, we identified 11 residues potentially involved in hapten binding and mutated them individually to alanines. The binding constant of each mutant was determined by fluorescence titration, and each mutant was tested individually as well as in competitive SIP experiments for infectivity. After three rounds of SIP, only molecules with KD values within a factor of 2 of the tightest binder remain, and among those, a mutant no longer carrying an unnecessary exposed tryptophan residue is preferentially selected. SIP is shown to select for the best overall properties of the displayed molecules, including folding behavior, stability and affinity.
Pedrazzi
G
G
Biochemisches Institut, Universität Zürich, Switzerland.
Schwesinger
F
F
Honegger
A
A
Krebber
C
C
Plückthun
A
A
D000906:Antibodies / Q000737:chemistry / Q000276:immunology*; D000915:Antibody Affinity; D000937:Antigen-Antibody Reactions; D003090:Coliphages / Q000235:genetics / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics / Q000821:virology; D016650:Fluorescein-5-isothiocyanate; D006241:Haptens / Q000737:chemistry / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D010957:Plasmids; D017510:Protein Folding; D013050:Spectrometry, Fluorescence
D000906:Antibodies; D006241:Haptens; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; C055966:immunoglobulin Fv; D016650:Fluorescein-5-isothiocyanate