A phage display technique for a fast, sensitive, and systematic investigation of protein-protein interactions.
Phage display is a technique in which a foreign protein or peptide is presented at the surface of a (filamentous) bacteriophage. This system, developed by Smith [(1985), Science 228, 1315-1317], was originally used to create large libraries of antibodies for the purpose of selecting those that strongly bound a particular antigen. More recently it was also employed to present peptides, domains of proteins, or intact proteins at the surface of phages, again to identify high-affinity interactions with ligands. Here we want to illustrate the use of phage display, in combination with PCR saturation mutagenesis, for the study of protein-protein interactions. Rather than selecting for mutants having high affinity, we systematically investigate the binding of every variant with its natural ligand. Via a modified ELISA we can calculate a relative affinity. As a model system we chose to display thymosin beta 4 on the phage surface in order to study its interaction with actin.
Rossenu
S
S
Flanders Interuniversity Institute for Biotechnology, Department of Biochemistry, Faculty of Medicine, University of Ghent, Belgium.
Dewitte
D
D
Vandekerckhove
J
J
Ampe
C
C
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D004797:Enzyme-Linked Immunosorbent Assay; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013947:Thymosin / Q000235:genetics / Q000378:metabolism*; D000099201:Thymosin beta(4)
D000199:Actins; D011506:Proteins; D013947:Thymosin; D000099201:Thymosin beta(4)