Expressed Protein Selenoester Ligation.

Area of Science:

• Biochemistry
• Chemical Biology
• Protein Chemistry

Background:

• Traditional protein semi-synthesis methods face limitations in efficiency and yield, especially for complex modifications or high dilution conditions.
• Intein-mediated protein ligation is a powerful tool, but requires optimization for diverse applications.
• Developing novel ligation strategies is crucial for advancing protein engineering and functional studies.

Purpose of the Study:

• To introduce a novel expressed protein selenoester ligation (EPSL) methodology for one-pot protein semi-synthesis.
• To demonstrate the efficiency and versatility of EPSL for producing modified proteins.
• To overcome limitations of traditional ligation methods, particularly at high dilution.

Main Methods:

• Development of expressed protein selenoester ligation (EPSL) using synthetic selenopeptides and protein aryl selenoesters.
• Generation of protein aryl selenoesters from expressed intein fusion precursors.
• In situ chemoselective deselenization for final protein formation.
• Application of EPSL for synthesizing ubiquitinated polypeptides, lipidated GTPase YPT6 analogues, and phosphorylated Hsp27 variants.

Main Results:

• EPSL enables one-pot semi-synthesis of modified proteins with high efficiency.
• The method is effective at high dilution concentrations, overcoming a key limitation of traditional techniques.
• Successful synthesis of diverse modified proteins, including ubiquitinated, lipidated, and phosphorylated variants, was achieved.

Conclusions:

• Expressed protein selenoester ligation (EPSL) is a robust and versatile methodology for protein semi-synthesis.
• EPSL offers significant advantages over traditional ligation methods, particularly for complex or dilute protein preparations.
• This technology facilitates the production of challenging protein constructs for further biological investigation.