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|Title:||Engineering of coiled-coil protein scaffolds as innovative tools for biosensing applications||Authors:||Gaiotto, Tiziano||Supervisore/Tutore:||Marzari, Roberto
|Cosupervisore:||Sblattero, Daniele||Issue Date:||8-Apr-2009||Publisher:||Università degli studi di Trieste||Abstract:||
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.
|Ciclo di dottorato:||XXI Ciclo||metadata.dc.subject.classification:||NANOTECNOLOGIE||Description:||
|Keywords:||biosensing tool; protein engineering; nanotechnological applications||Type:||Doctoral||Language:||en||Settore scientifico-disciplinare:||BIO/06 ANATOMIA COMPARATA E CITOLOGIA||NBN:||urn:nbn:it:units-7426|
|Appears in Collections:||Scienze biologiche|
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