Structure-based engineering and comparison of novel split inteins for protein ligation

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Protein splicing involves intein-mediated excision and extein ligation.
• Split inteins facilitate protein trans-splicing for ligating separate polypeptide chains.
• Existing split inteins have cross-reactivity issues, limiting applications.

Purpose of the Study:

• To develop novel split inteins for efficient and orthogonal protein ligation.
• To expand the toolkit for biotechnological applications like protein semi-synthesis and engineering.

Main Methods:

• Derived 21 new split inteins from four efficient cis-splicing inteins.
• Systematically compared trans-splicing efficiency of 24 split inteins.
• Tested cross-activities among split intein fragments to identify orthogonal pairs.

Main Results:

• Successfully derived 21 novel split inteins.
• Identified orthogonal split intein fragments suitable for multiple-ligation applications.
• Demonstrated potential for enhanced protein ligation in biotechnological contexts.

Conclusions:

• The newly developed split inteins offer improved orthogonality and efficiency for protein ligation.
• These novel reagents expand possibilities for advanced protein engineering and chemical biology.
• This work addresses limitations of current split inteins, paving the way for broader applications.