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Engineering protein theranostics using bio-orthogonal asparaginyl peptide ligases
- Source :
- Theranostics
- Publication Year :
- 2021
-
Abstract
- Background: Protein theranostics integrate both diagnostic and treatment functions on a single disease-targeting protein. However, the preparation of these multimodal agents remains a major challenge. Ideally, conventional recombinant proteins should be used as starting materials for modification with the desired detection and therapeutic functionalities, but simple chemical strategies that allow the introduction of two different modifications into a protein in a site-specific manner are not currently available. We recently discovered two highly efficient peptide ligases, namely butelase-1 and VyPAL2. Although both ligate at asparaginyl peptide bonds, these two enzymes are bio-orthogonal with distinguishable substrate specificities, which can be exploited to introduce distinct modifications onto a protein. Methods: We quantified substrate specificity differences between butelase-1 and VyPAL2, which provide orthogonality for a tandem ligation method for protein dual modifications. Recombinant proteins or synthetic peptides engineered with the preferred recognition motifs of butelase-1 and VyPAL2 at their respective C- and N-terminal ends could be modified consecutively by the action of the two ligases. Results: Using this method, we modified an EGFR-targeting affibody with a fluorescein tag and a mitochondrion-lytic peptide at its respective N- and C-terminal ends. The dual-labeled protein was found to be a selective bioimaging and cytotoxic agent for EGFR-positive A431 cancer cells. In addition, the method was used to prepare a cyclic form of the affibody conjugated with doxorubicin. Both modified affibodies showed increased cytotoxicity to A431 cells by 10- and 100-fold compared to unconjugated doxorubicin and the free peptide, respectively. Conclusion: Bio-orthogonal tandem ligation using two asparaginyl peptide ligases with differential substrate specificities is a straightforward approach for the preparation of multifunctional protein biologics as potential theranostics. Ministry of Education (MOE) Published version This research was supported by Academic Research Grant Tier 3 (MOE2016-T3-1-003) from the Singapore Ministry of Education (MOE) to J.P.T., J. L., and C.-F. L.
- Subjects :
- bio-orthogonal ligation
Protein Labelling
Protein Theranostics
Medicine (miscellaneous)
Peptide
Conjugated system
Protein Engineering
010402 general chemistry
01 natural sciences
protein labelling
Substrate Specificity
law.invention
Ligases
03 medical and health sciences
law
Cell Line, Tumor
protein theranostics
Humans
Peptide bond
Precision Medicine
Pharmacology, Toxicology and Pharmaceutics (miscellaneous)
030304 developmental biology
chemistry.chemical_classification
0303 health sciences
peptide asparaginyl ligases
Protein engineering
Recombinant Proteins
Mitochondria
0104 chemical sciences
ErbB Receptors
Enzyme
chemistry
Biochemistry
Doxorubicin
Biological sciences::Biochemistry [Science]
Cancer cell
MCF-7 Cells
Recombinant DNA
Peptides
Protein Processing, Post-Translational
A431 cells
Research Paper
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Journal :
- Theranostics
- Accession number :
- edsair.doi.dedup.....4637a4dbd5e9485f140c63b0bca8ea03