pH-Controlled Protein Orthogonal Ligation Using Asparaginyl Peptide Ligases

Area of Science:

• Biochemistry
• Protein Engineering
• Chemical Biology

Background:

• Peptide asparaginyl ligases (PALs) are known for efficient peptide ligation at asparaginyl (Asn) residues.
• PALs typically require neutral pH for Asn ligation and acidic pH for aspartyl (Asp) ligation, limiting Asp-specific ligation efficiency.
• The use of PALs for Asp-specific ligation has been underexplored due to perceived inefficiencies at acidic pH.

Purpose of the Study:

• To investigate and optimize the use of PAL enzymes for efficient Asp-specific peptide ligation.
• To develop a pH-controlled orthogonal ligation strategy utilizing PALs for sequential labeling.
• To demonstrate the application of this method in creating dual-labeled protein biologics.

Main Methods:

• Assessed catalytic activity of PAL enzymes, including VyPAL2, toward P1-Asp substrates at acidic pH (4-5).
• Compared PALs' activity with sortase A for Asp ligation.
• Developed and applied a sequential, pH-controlled ligation strategy for dual labeling of proteins (sfGFP and affibody).

Main Results:

• PAL enzymes exhibit significantly higher catalytic activity toward P1-Asp substrates at pH 4-5 compared to sortase A.
• Achieved efficient intra- and intermolecular ligations at Asp junctions using PALs under acidic conditions.
• Successfully performed sequential N-to-C and C-to-N dual labeling of proteins with distinct payloads (peptides, drugs, dyes).

Conclusions:

• PAL enzymes demonstrate practical utility for Asp-specific ligation at acidic pH, overcoming previous limitations.
• pH-controlled orthogonal ligation using PALs enables precise sequential modification of proteins.
• This methodology facilitates the preparation of sophisticated, multifunctional protein biologics for pharmacological applications.