Engineering protein theranostics using bio-orthogonal asparaginyl peptide ligases

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Protein theranostics combine diagnostic and therapeutic functions but face challenges in multimodal agent preparation.
• Current methods lack site-specific strategies for dual modifications on conventional recombinant proteins.
• Two newly discovered peptide ligases, butelase-1 and VyPAL2, offer bio-orthogonal and distinguishable substrate specificities.

Purpose of the Study:

• To develop a straightforward method for site-specific dual modification of proteins using bio-orthogonal tandem ligation.
• To exploit the differential substrate specificities of butelase-1 and VyPAL2 for creating multifunctional protein biologics.
• To demonstrate the utility of this method for preparing potential cancer theranostics.

Main Methods:

• Quantified substrate specificity differences between butelase-1 and VyPAL2 to establish orthogonality.
• Engineered recombinant proteins with specific recognition motifs for sequential modification by the two ligases.
• Applied tandem ligation to an EGFR-targeting affibody for dual labeling with a fluorescein tag and a mitochondrion-lytic peptide.

Main Results:

• Successfully created a dual-labeled affibody with selective bioimaging and cytotoxic activity against EGFR-positive A431 cancer cells.
• Prepared a cyclic affibody conjugated with doxorubicin, demonstrating enhanced cytotoxicity.
• Achieved a 10-fold and 100-fold increase in cytotoxicity compared to unconjugated doxorubicin and the free peptide, respectively.

Conclusions:

• Bio-orthogonal tandem ligation using two asparaginyl peptide ligases with differential specificities is an effective approach for preparing multifunctional protein biologics.
• This method provides a straightforward strategy for developing novel protein-based theranostics.
• The developed technique holds significant potential for advancing targeted cancer therapy and diagnostics.