Please use this identifier to cite or link to this item: https://hdl.handle.net/10356/148023
Title: Engineering protein theranostics using bio-orthogonal asparaginyl peptide ligases
Authors: Wang, Zhen
Zhang, Dingpeng
Hemu, Xinya
Hu, Side
To, Janet
Zhang, Xiaohong
Lescar, Julien
Tam, James P.
Liu, Chuan-Fa
Keywords: Science::Biological sciences::Biochemistry
Issue Date: 2021
Source: Wang, Z., Zhang, D., Hemu, X., Hu, S., To, J., Zhang, X., Lescar, J., Tam, J. P. & Liu, C. (2021). Engineering protein theranostics using bio-orthogonal asparaginyl peptide ligases. Theranostics, 11(12), 5863-5875. https://dx.doi.org/10.7150/thno.53615
Project: MOE2016-T3-1-003
Journal: Theranostics
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.
URI: https://hdl.handle.net/10356/148023
ISSN: 1838-7640
DOI: 10.7150/thno.53615
Rights: © 2021 The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions.
Fulltext Permission: open
Fulltext Availability: With Fulltext
Appears in Collections:SBS Journal Articles

Files in This Item:
File Description SizeFormat 
Engineering protein theranostics using bio-orthogonal asparaginyl peptide ligases.pdf2.23 MBAdobe PDFView/Open

Page view(s)

34
Updated on Sep 21, 2021

Download(s)

10
Updated on Sep 21, 2021

Google ScholarTM

Check

Altmetric


Plumx

Items in DR-NTU are protected by copyright, with all rights reserved, unless otherwise indicated.