Site-Specific Sequential Protein Labeling Catalyzed by a Single Recombinant Ligase.

Area of Science:

• Biochemistry
• Protein Engineering
• Molecular Biology

Background:

• Protein ligases are crucial for protein engineering but often lead to product hydrolysis.
• Sequential protein labeling requires orthogonal ligases due to substrate recognition limitations.

Purpose of the Study:

• To develop a novel protein ligation strategy using a promiscuous enzyme.
• To enable efficient and sequential site-specific protein modification.

Main Methods:

• Utilized a recombinant Oldenlandia affinis asparaginyl endopeptidase (OaAEP1) with promiscuous nucleophile acceptance.
• Designed a ligation module that is efficiently ligated but poorly recognized by OaAEP1.
• Demonstrated sequential N- and C-terminal modification of a single protein.

Main Results:

• Achieved efficient ligation with a defined product that resists further hydrolysis.
• Showcased the ability to sequentially modify both N- and C-termini of a substrate protein.
• Obtained excellent yields in sequential protein modifications.

Conclusions:

• The developed method offers a versatile tool for site-specific protein engineering.
• This approach overcomes limitations of existing protein ligation techniques.
• Enables precise control over protein modification for advanced applications.