Use of bifunctional hybrid beta-lactamases for epitope mapping and immunoassay development.
Mapping of epitopes is a crucial step for the study of immune pathways, the engineering of vaccines and the development of immunoassays. In this work, the Bacillus licheniformis beta-lactamase BlaP has been engineered to display heterologous polypeptides in a permissive and solvent-exposed loop. When combined with phage display, this modified enzyme can be used for epitope mapping by cloning random gene fragments. The procedure presented in this paper allows the selection of large infectious phage libraries with high diversity and efficient beta-lactamase activities. A useful aspect of the proposed technique results from the possibility of using the beta-lactamase activity carried by phages to evaluate the proportion of immobilised phages during the successive enrichment steps of the library or competition experiments with the selected phages. Another advantage of the technique derives from the fact that the epitope is selected as a bifunctional hybrid protein, which can be overproduced and purified. The resulting recombinant protein associates an epitope with a specific and efficient enzymatic activity. This constitutes an original tool for immunoassay development. A virus influenza hemagglutinin (HA1)-gene fragment library has been generated with this system and used to identify a linear epitope.
Chevigné
Andy
A
Macromolécules Biologiques, Centre d'Ingénierie des Protéines, Institut de Chimie B6a, Université de Liège, Sart-Tilman, B4000 Liège, Belgium.
Yilmaz
Nursel
N
Gaspard
Gilles
G
Giannotta
Fabrizio
F
François
Jean Marie
JM
Frère
Jean Marie
JM
Galleni
Moreno
M
Filée
Patrice
P
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics; D020134:Catalytic Domain; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000276:immunology; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D007118:Immunoassay / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments; D019151:Peptide Library; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D001618:beta-Lactamases / Q000276:immunology*
D000939:Epitopes; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D010446:Peptide Fragments; D019151:Peptide Library; C493831:hemagglutinin HA1, influenza B virus; D001618:beta-Lactamases