The Study Of Chemical Synthesis Of Polyubiquitin And Ubiquitin Like Proteins

Ubiquitination and ubiquitin like modifications widely exist in eukaryotic cells,and are involved in a wide range of cellular processes,including signal transduction,damage repairing of DNA and degradation of intracellular proteins.Ubiquitination and ubiquitin like modifications of different substrates are diverse and have different functions,which ensure the diversity and specificity of the modification functions.At present,a considerable part of the ubiquitin and ubiquitin like proteins with important functions are not readily available due to the lack of specific enzymatic synthesis systems.Meanwhile,it is more difficult to obtain a purely homogeneous ubiquitin probe to study the hydrolysis mechanism of DUBs.Protein chemical synthesis technology,by virtue of its ability to precisely construct proteins at the atomic scale,provides an effective strategy in the case of conventional biochemical enzyme constraints.In this paper,the chemical synthesis of some important ubiquitin and ubiquitin like proteins were investigated by protein chemical synthesis technology.The ubiquitin like protein NEDD8 and ubiquitin probe Ub-AMC were successfully synthesized with fewer steps and higher efficiency compared with previous work by the one-pot two-segment ligation-desulfurization strategy developed by our group.Taking advantage of semisynthesis method based on unnatural amino acids site-specific incorporation technology,the study of the preparation of polyubiquitin and polyubiquitin like proteins were also carried out.We successfully obtained an ubiquitin fragment that can be used for ligation reaction by incorporating Alloc-Lys and using Boc to protect other Lys side-chain amino groups.And the auxiliary mediated native chemical ligation were applied as well to achieve efficient preparation of the isopeptide bond between two ubiquitin segement.The successful preparation of natural diubiquitin containing isopeptide bond at K48 site verifies the feasibility of the above strategy.This work provides a universal approach for the synthesis of polyubiquitin and ubiquitin like proteins of other sites by chemical method,and lays the foundation for the further study of ubiquitination,ubiquitin like modifications specific recognition and mechanism of degradation process.