Generation of von Willebrand factor epitope libraries expressed in E. coli.
The von Willebrand factor (VWF) subunit is composed of several domains, often coinciding with structural regions, characterized through their specific interaction with a ligand. Since several monoclonal antibodies (MoAbs) have been shown to functionally interfere with one of the specific interactions, we have created libraries of bacterial clones expressing peptidic sequences of VWF to map antibodies directed against this protein. Randomly cleaved fragments of VWF cDNA have been cloned in a plasmid designed for the expression of small peptides as part of larger fusion proteins. The NovaTope system is a useful procedure for protein analysis, allowing screening of epitopes composed of contiguous amino acid residues. To map MoAbs with conformational discontinuous epitopes displayed on small as well as large peptidic domains, this technique had to be widely modified to obtain two VWF peptide libraries expressing two ranges of peptide length (15-70 and 100-300 amino acids). Screening with six MoAbs with an epitope in a known region was performed to control both libraries. Four MoAbs were mapped through the characterization of overlapping sequences for 5-10 different positively expressed clones respectively. Two of these mapped MoAbs had no known inhibitory effect and bind reduced VWF only. The fact that the two other MoAbs mapped VWF functional interactions with ligands, platelet GPIIb/IIIa and Factor VIII, respectively, demonstrate that our libraries are valuable tools to determine conformational epitopes.
Jumilly
A L
AL
Inserm U 143, Hĵpital de Bicêtre, Paris, France.
Houllier
A
A
Meyer
D
D
Girma
J P
JP
Kerbiriou-Nabias
D
D
D000911:Antibodies, Monoclonal / Q000276:immunology; D003001:Cloning, Molecular / Q000379:methods*; D018985:Epitopes, B-Lymphocyte / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics*; D015723:Gene Library; D017422:Sequence Analysis, DNA; D014841:von Willebrand Factor / Q000235:genetics* / Q000276:immunology
D000911:Antibodies, Monoclonal; D018985:Epitopes, B-Lymphocyte; D014841:von Willebrand Factor