טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentEid Emad
SubjectTotal Chemical Synthesis of SUMO-2-Lys63-linked
Di- ubiquitin Hybrid Chains Assisted by Removable
Solubilizing Tags
DepartmentDepartment of Chemistry
Supervisor Professor Ashraf Brik
Full Thesis textFull thesis text - English Version


Abstract

Post translational modifications (PTMs) have a profound impact on the structure, function, and localization of the modified proteins. PTMs can be found in the form of a small or large molecule or even a small protein such as the case of the Small Ubiquitin Like Modifier (SUMO) proteins which are known to regulate many important cellular processes such as: transcription, response to stress and nuclear cytosolic transport.

Recently, it has been found that SUMO-ubiquitin hybrid chains containing SUMO-2 linked to Lys63-di-ubiquitin play a major role in DNA repair. Despite progress in understanding the role of these hybrid chains in DNA repair, there are still many fundamental questions remained to be answered. The first step in the investigation of the importance of these chains, and their role in DNA damage repair would be the preparation of homogeneous materials of SUMO-ubiquitin hybrid chains and their different analogues in workable quantities. Reported here is the first total chemical synthesis of four different SUMO-2-Lys63-linked di-ubiquitin hybrid chains, in which the di-ubiquitin is attached through different lysines of SUMO. The synthesis of these chains was achieved by applying the current state of the art chemical methods for protein synthesis, where the usefulness of removable solubilizing tags was demonstrated as well. Two different approaches were examined in terms of reliability and efficiency. In the first approach, a poly-Arg tag was attached to the C-terminus of SUMO via a 3,4-diaminobenzloxy cleavable linker, while in the second the tag was attached through the newly developed Amino Phenylactamidomethyl linker, which can be cleaved by PdCl2.

The results of this comparison between these two approaches, draw new principles that would be useful when dealing with challenging proteins and assists in overcoming the different difficulties in their synthesis. Moreover, it offers guidelines for future scale-up preparation of these analogues for future biochemical, biophysical, and functional analyses.