Encouraging Solution to the Problem of Synthesizing Protein α‐Thioester

Abstract

Comprehensive SummaryExpressed 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 protein α‐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).

Multifunctional Lipidated Protein Carrier with a Built-In Adjuvant as a Universal Vaccine Platform Potently Elevates Immunogenicity of Weak Antigens

“…N -terminal transamination reaction reported by Francis et al. provides a straightforward and efficient method for modifying ketone or aldehyde functional groups in a specific location. − By employing this strategy, Pyridoxal 5′-Phosphate (PLP) has been successfully utilized to modify various proteins, forming an aldehyde or ketone functional group at their N -terminus, which can further react with alkoxyamine compounds, resulting in the formation of oxime-linked structures . Importantly, this site-specific conjugation strategy utilizes extremely gentle reaction conditions, making it well-suited for meeting extensive production needs.…”