Expressed protein ligation(EPL)provides a powerful tool to access large-size proteins with precise structures.Existing methods for constructing the critical protein thioester for EPL have predominantly relied on the r...Expressed protein ligation(EPL)provides a powerful tool to access large-size proteins with precise structures.Existing methods for constructing the critical protein thioester for EPL have predominantly relied on the recombinant intein fusion expressed in Escherichia coli(E.coli).Despite its powerful applications,the expression of thioester derived from eukaryotic protein in E.coli inherently suffers from its limited solubility,the inactivity of intein,premature hydrolysis and low yields.To overcome these obstacles,we present herein the facile one-flask synthesis of inaccessible proteinα-thioester via a SUMO-protein-intein(SPI)sandwich model.The utility of SUMO enhances the protein fusion yield and solubility,prevents premature hydrolysis and simplifies the purification process.The inaccessible protein thioester with internal Cys residues can be readily produced and is compatible with the EPL-desulfurization protocol used to prepare complex proteins,which is otherwise difficult to obtain using traditional methods.Its utility has been highlighted through the synthesis of human granulocyte colony-stimulating factor(G-CSF).展开更多
基金supported by the National Science Fund for Distinguished Young Scholars(22225701)the Interdisciplinary Program of Shanghai Jiao Tong University(20230102)+3 种基金the National Natural Science Foundation of China(22077080,92253302 and 22275122)the Shanghai Pilot Program for Basic Research Shanghai Jiao Tong University(21TQ1400210)the Special Projects of the Central Government in Guidance of Local Science and Technology Development(2021Szvup077)the China Post-doctoral Science Foundation(2023M732214).
文摘Expressed protein ligation(EPL)provides a powerful tool to access large-size proteins with precise structures.Existing methods for constructing the critical protein thioester for EPL have predominantly relied on the recombinant intein fusion expressed in Escherichia coli(E.coli).Despite its powerful applications,the expression of thioester derived from eukaryotic protein in E.coli inherently suffers from its limited solubility,the inactivity of intein,premature hydrolysis and low yields.To overcome these obstacles,we present herein the facile one-flask synthesis of inaccessible proteinα-thioester via a SUMO-protein-intein(SPI)sandwich model.The utility of SUMO enhances the protein fusion yield and solubility,prevents premature hydrolysis and simplifies the purification process.The inaccessible protein thioester with internal Cys residues can be readily produced and is compatible with the EPL-desulfurization protocol used to prepare complex proteins,which is otherwise difficult to obtain using traditional methods.Its utility has been highlighted through the synthesis of human granulocyte colony-stimulating factor(G-CSF).
