- Engineering of coiled-coil protein scaffolds as innovative tools for biosensing applications
- Gaiotto, Tiziano
Subject
- biosensing tool
- protein engineering
- nanotechnological applications
- NANOTECNOLOGIE
- BIO/06 ANATOMIA COMPARATA E CITOLOGIA
Description
- 2007/2008
- A new generation of protein scaffolds is becoming a valid alternative tool to recombinant antibodies of biotechnological, medical and pharmaceutical applications, where strong affinity and specificity are required. They share with antibodies important features (target affinity and specificity), but they have also some improvements (smaller size of molecule, tolerance to modification of the framework and the recognition site restricted to few residues), that can be exploited for biosensing application in nanotechnological platforms. Nanotechnology has been played an increasingly important role in the development of biosensors, improving the intrinsic features of biodevices. In this thesis work, we analyzed the coiled-coil domain, a widely spread dimerization domain shared by several protein scaffolds, and involved in protein-protein interaction in both eukaryotic and prokaryotic cells. The analysis of the coiled-coil structure allows a de novo design of new peptides, namely E and K, that can dimerize as a E/K coiled-coil system: the dimerization feature and the stability of the interaction makes this system an ideal platform to build up functional and customizable biosensors. A characterization of the E/K interaction was performed by using the protein complementation assay (PCA), a useful biological method to investigate the interaction between protein partners. With this in vivo method, we corroborate the interaction features determinate with circular dichroism, and we demonstrated that E and K coils effectively represent a protein scaffold, able to tolerate amino acid substitutions without altering its main structure. In addition, we create two libraries of K mutant coils, randomizing the peptide sequence, and with PCA we selected new K binders (Kran 5.17 and Krd F8) that showed a comparable interaction activity with the E-coil in preliminary in vitro tests. In the last part of this work, we generate a library of a new scaffold molecule (the single chain E-K) capable to bind small molecules as a single protein product containing both domains. Using the phage display selection system, we isolated scsE-K that can bind our analyte (the caffeine) with high specificity. This new molecules can be a powerful tool for analytical and biomedical applications.
- XXI Ciclo
- 1980
Date
- 2009-04-30T12:59:19Z
- 2009-04-30T12:59:19Z
- 2009-04-08
Type
- Doctoral Thesis
Format
- application/pdf
Identifier
urn:nbn:it:units-7426