Zur Kurzanzeige

dc.contributor.author
Harmand, Thibault J.R.
dc.contributor.supervisor
Bode, Jeffrey W.
dc.contributor.supervisor
Hilvert, Donald
dc.date.accessioned
2017-11-17T07:08:00Z
dc.date.available
2017-11-16T21:57:15Z
dc.date.available
2017-11-17T07:08:00Z
dc.date.issued
2017
dc.identifier.uri
http://hdl.handle.net/20.500.11850/209686
dc.identifier.doi
10.3929/ethz-b-000209686
dc.description.abstract
Proteins are involved in and perform a myriad of biological functions. Understanding their structures, functions and interactions with small molecules or other proteins is essential for modern day science. In recent years, proteins as therapeutics have become more and more relevant in the pharmaceutical industry, because of their low toxicity, low risk of side effects as well as their high specificity. Hence, obtaining pure and homogenous samples of proteins has become an important field of research. Although recombinant production allows access to a large number of proteins, it does present limitations: incorporation of unnatural amino acids and the highly sought after posttranslational modifications (PTMs) – that are often critical for the biological activity of the protein of interest – are difficult, if not impossible, to implement. Chemical protein synthesis appears to be a good alternative to the recombinant approach, as it can easily bypass the mentioned limitations. It can deliver homogenous protein samples with almost any kind of amino acid component, without biological contaminants. In 2006, the Bode group developed a method called the -ketoacid–hydroxylamine ligation (KAHA ligation) which allows the chemical synthesis of proteins by ligating two unprotected peptide segments. The KAHA ligation proceeds via the decarboxylative condensation of an -ketoacid with a hydroxylamine to form an amide bond under acidic organic buffering conditions at slightly elevated temperature. Since its discovery, the KAHA ligation has been subject to constant development, allowing the Bode group to synthesize proteins like SUMO2, SUMO3, irisin, AS48, NP4, betatrophin, interleukin-2 proteins and IFITM3. This dissertation describes, in the first chapter, the synthetic improvements made for two main building blocks: the cyanosulfur ylide linker and the (S)-N-Boc-5-oxaproline, used for the preparation of -ketoacid peptide segments and hydroxylamine peptide segments respectively. The new procedures enable the production of these monomers on a multigram scale, allowing our group to scale up the synthesis of synthetic proteins. The second chapter describes the usage of the KAHA ligation for the preparation of multimilligram quantities of the protein hormone betatrophin. This protein had attracted attention after primary results claimed it had direct effects on the proliferation of -cells, giving rise to hope for a new approach to treat diabetes. This synthesis represents the first report of a synthetic protein assembled via five peptide segments by KAHA ligation. Integral membrane proteins are, because of their hydrophobicity, challenging to obtain both by recombinant overexpression and chemical synthesis. In a third chapter, the KAHA ligation was used to synthesize one of these challenging molecules: the antiviral transmembrane protein IFITM3. The unique features of the KAHA ligation – acidic organic solvents and the formation of a depsi-peptide at the ligation site – appeared to be ideally suited for the synthesis of such hydrophobic proteins, and we could successfully synthesize the desired protein in a multimilligram quantity. A phosphorylated, a palmitoylated and a fluorescent variant of IFITM3 could also be synthesized using the KAHA ligation. We showed that the synthetic IFITM3 was incorporating itself into a lipid membrane and displaying an alpha helix fingerprint, as predicted by the calculated 3D structure. Preliminary results using lipid vesicles and fluorescently labeled viruses showed that our synthetic material displays antiviral activity and more experiments are currently being conducted.
en_US
dc.language.iso
en
en_US
dc.publisher
ETH Zurich
en_US
dc.rights.uri
http://rightsstatements.org/page/InC-NC/1.0/
dc.subject
Betatrophin
en_US
dc.subject
IFITM3
en_US
dc.subject
Chemical protein synthesis
en_US
dc.subject
KAHA ligation
en_US
dc.subject
Membrane proteins
en_US
dc.subject
Protein modifications
en_US
dc.title
Chemical Synthesis of Betatrophin and the Integral Membrane Protein IFITM3 by KAHA Ligation
en_US
dc.type
Doctoral Thesis
dc.rights.license
In Copyright - Non-Commercial Use Permitted
dc.date.published
2017-11-17
ethz.size
206 p.
en_US
ethz.code.ddc
5 - Science::540 - Chemistry
en_US
ethz.grant
Protein Synthesis with Chemoselective Ligations
en_US
ethz.grant
Chemoselective Protein Ligation
en_US
ethz.identifier.diss
24309
en_US
ethz.publication.place
Zurich
en_US
ethz.leitzahl
ETH Zürich::00002 - ETH Zürich::00012 - Lehre und Forschung::00007 - Departemente::02020 - Dep. Chemie und Angewandte Biowiss. / Dep. of Chemistry and Applied Biosc.::02514 - Laboratorium für Organische Chemie / Laboratory of Organic Chemistry::03861 - Bode, Jeffrey W. / Bode, Jeffrey W.
en_US
ethz.grant.agreementno
169451
ethz.grant.agreementno
150073
ethz.grant.fundername
SNF
ethz.grant.fundername
SNF
ethz.grant.funderDoi
10.13039/501100001711
ethz.grant.funderDoi
10.13039/501100001711
ethz.grant.program
Projektförderung in Mathematik, Natur- und Ingenieurwissenschaften (Abteilung II)
ethz.grant.program
ethz.date.deposited
2017-11-16T21:57:17Z
ethz.source
FORM
ethz.eth
yes
en_US
ethz.availability
Open access
en_US
ethz.rosetta.installDate
2017-11-17T07:08:17Z
ethz.rosetta.lastUpdated
2018-11-06T02:01:25Z
ethz.rosetta.exportRequired
true
ethz.rosetta.versionExported
true
ethz.COinS
ctx_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.atitle=Chemical%20Synthesis%20of%20Betatrophin%20and%20the%20Integral%20Membrane%20Protein%20IFITM3%20by%20KAHA%20Ligation&rft.date=2017&rft.au=Harmand,%20Thibault%20J.R.&rft.genre=unknown&rft.btitle=Chemical%20Synthesis%20of%20Betatrophin%20and%20the%20Integral%20Membrane%20Protein%20IFITM3%20by%20KAHA%20Ligation
 Suchen via SFX

Dateien zu diesem Eintrag

Thumbnail

Publikationstyp

Zur Kurzanzeige