Exploration Of Hydrazide-based Protein Synthesis Methods

Chemical protein synthesis starts from simple protected amino acids and employs solid-phase peptide synthesis,recombinant protein expression and solution peptide ligation chemistry to achieve in vitro bioorganic synthesis.This approach makes it possible to construct the primary structures of proteins at atomic level,which allows incorporation of unnatural amino acid residues and offers higher synthetic flexibility and selectivity compared with bioconjugation and amber suppression technology.Native chemical ligation represents the most popular method of chemical protein synthesis.Recently,our group found that peptide hydrazides can be valuable substrates for ligations.To explore the practical utility of peptide hydrazides,the author describes some methodological progress of hydrazide-based protein synthesis in this dissertation.The first part explores the combinatorial use of hydrazide-based chemical ligation with expressed protein ligation.The author chose hydrophilic globular protein LC3 and hydrophobic lipoprotein LC3-? as model targets.To assemble LC3-? through semi-synthesis,the author developed a new side-chain solubilizing tagging strategy.The side-chain polyarginine tag ensured sufficient solubility of protein segments and was removable under mild ultraviolet irradiation.This strategy permits detergent-free synthesis of lipoproteins for the first time,which can streamline the otherwise cumbersome synthesis protocol.It is expected to be robust and useful for the preparation of other hydrophobic peptides and proteins.The second part focuses on the generation and reaction of peptide hydrazides.Methodological advances include solid-phase synthesis of peptide hydrazides using new generation hydrazide resins and recombinant expression of peptide hydrazides using intein technology or hydroxy acid incorporation technology.The versatility of sources of peptide hydrazides will further extend the flexibility of hydrazide-based chemical ligation.The author also discovered a new thiol catalyst methyl thioglycolate in the ligation,which can assist one-pot two-segment or three-segment ligation/desulfurization reactions to synthesize proteins more efficiently.The protocol was employed to generate crystalline monoubiquitin on a 30-milligram scale.The new catalyst is potent and cheap,which is of potential use in large-scale chemical synthesis of high-purity protein molecules.