Synthesis of Sonic Hedgehog protein and development of peptide binders using phage display libraries
Chemical protein synthesis has become a potent tool for the synthesis of proteins, especially those which are not easily accessed by DNA recombinant methods, such as proteins with posttranslational modifications. The key reaction in chemical protein synthesis is Native Chemical Ligation, which impli...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2018 |
| País: | España |
| Institución: | CBUC, CESCA |
| Repositorio: | TDR. Tesis Doctorales en Red |
| OAI Identifier: | oai:www.tdx.cat:10803/552403 |
| Acceso en línea: | http://hdl.handle.net/10803/552403 |
| Access Level: | acceso abierto |
| Palabra clave: | Síntesi proteica Síntesis de proteínas Protein synthesis Pèptids Péptidos Peptides Ciències Experimentals i Matemàtiques 547 |
| Sumario: | Chemical protein synthesis has become a potent tool for the synthesis of proteins, especially those which are not easily accessed by DNA recombinant methods, such as proteins with posttranslational modifications. The key reaction in chemical protein synthesis is Native Chemical Ligation, which implies a chemoselective ligation between two unprotected peptide fragments in aqueous solution. It requires a cysteine residue at the N-terminal of one peptide, and a thioester moiety at the C-terminal of the other fragment. Several strategies have been developed for the synthesis of C-terminal thioester peptides using Fmoc-SPPS. In our group thioester peptides are obtained following the N-acylurea approach, developed by Blanco- Canosa and Dawson, which gives N-acyl-N’-methylacylurea (MeNbz) peptides as precursors of thioesters. However, this approach just enables the ligation of peptides on the C to N direction. In the last years, other strategies using masked or cryptothioesters used for the synthesis of proteins by KCL have been reported. In the present thesis, we have developed new derivatives from the N-acylurea (MeDbz) linker which are acylated with p-cyanophenylchloroformate, rendering the p- cyannophenyloxycarbonyl-MeDbz peptides (abbreviated as pCN-Phoc-MeDbz). Upon activation under slightly basic conditions in solution, the linker cyclizes to render the corresponding MeNbz peptide, which in presence of thiols will suffer a thioesterification to finally yield the C-terminal thioester peptide able to undergo ligation. Essentially, this new linker derivative can act as a masked thioester and thereby, allows the presence of a N- terminal cysteine in the same peptide. We used this new methodology to perform Kinteic Controlled Ligations (KCL) in model peptides and successfully obtained the desired product. Moreover, we have demonstrated that the pCN-Phoc-MeDbz linker is orthogonal with the common reactions used during chemical proteins synthesis, such as desulfurization or Acm and Thz removal. This new methodology has been used for the synthesis of an analog of the Sonic Hedgehog protein (ShhN). The natural ShhN is a 174-mer lipoprotein with a palmitic residue at the N- terminal and a cholesterol molecule at the C-terminus. The interest of this protein relies on its important biological functions. ShhN is the main ligand on the Hedgehogh (Hh) signaling pathway, which is implicated in patterning during the embryo development. In adults, the Hh regulates the cell growth, and it is implied in the maintenance of stem cells and also in tissue regeneration. Aberrant activity on this pathway has been related to several types of cancer, and the majority are ligand dependent. Although the importance of ShhN in this aberrant activation, most of the inhibitors of the Hh pathway target downstream components of the signaling cascade. Actually, only two molecules, are known to bind ShhN. The main goal of the thesis was to synthetize the natural ShhN and also some analogs that could be used for further biological studies such as phage display in order to find ligands to the protein. We applied the methodology developed to chemically synthetize the natural ShhN and two analogues. Unfortunatelly, due to the high hydrophobic character of the natural ShhN, we could not obtain the natural protein. Nevertheless, we succeeded on the synthesis of two analogs of ShhN. On one side, we synthetized Palm-ShhN-biotin, which maintained the natural palmitic acid at the N-temrinus, but changed the cholesterol moiety at the C-terminus by a biotin. On the other side, an analog harboring an Ile dipeptide at the N-terminus and a biotin at the C-terminus was also prepared. After peptide assembly to achieve the final ShhN analogues, they were properly folded to undergo further biological studies. The next stage on our project was to find binders to the synthetic ShhN analogs. To this end, we envisaged to use phage display technology to possibly find new peptide binders to ShhN analogs. This technique allows the winnowing down from libraries with high diversity, up to 109, to finally obtain few binders. Although the first encountered ligands may have high Kd values, affinity maturation allows the obtainment of tighter binders derived from the first selected. We carried out a research stay at Professor Sidhu’s lab, who is an expert in phage display. There, we screened eight scaffold peptide libraries against the synthetic targets. Although the libraries were completely naïve for our target, we obtained few binders. And fortunately, after performing affinity assays, the estimate IC50 of the binders were 40 nM. Future work will be focused on the construction of affinity maturation libraries and on the validation of the affinity of the selected binders off phage. |
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