Artificial "antibodies" via combinatorial protein design
Skerra, A., Beste, G., Schmidt, F., Stibora, T.
Institut für Biochemie, Technische Universität, Petersenstr. 22, D-64287 Darmstadt, Germany
During the past 100 years antibodies have been considered as ideal tools for molecular recognition. Especially with the methods for the bacterial production of their fragments attempts have furthermore been made to develop improved antibodies by protein engineering. However, immunoglobulins may not be optimally suited for modern applications because of their composition of two polypeptide chains and the comparably large size even of their Fv fragment. The lipocalins, a family of small proteins whose first member was the serum retinol-binding protein (RBP), could offer an alternative protein architecture for the binding of prescribed ligands. Despite low sequence homology they share a structurally conserved beta-barrel, which supports four loops at one end that form the entrance to the binding pocket. The loops exhibit large conformational differences between individual lipocalins and give rise to the variety of natural ligand specificities. The architecture is thus reminiscent of immunoglobulins with their hypervariable loops on top of a rigid framework. In a combinatorial protein design approach the bilin-binding protein (BBP) was used in order to create lipocalin variants that recognize novel ligands. 16 amino acids in the four loops were subjected to targeted random mutagenesis. The library with 3.7 x 108 mutants was employed for panning with different immobilized compounds by phage display. In the case of fluorescein mutants with Kd values as low as 37 nM were obtained. Lipocalin variants with specificities for other organic molecules and peptides have been generated as well. We therefore term these new ligand-binding proteins, which may substitute conventional antibodies in several instances, as "anticalins".
| LOCATION |
DATE |
TIME |
| Lecture Hall II |
Sunday, April 5 |
06:40 pm |