Enhancing Cellular Uptake of Native Proteins through Bio-Orthogonal Conjugation with Chemically Synthesized Cell-Penetrating Peptides
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture Maintenance
2.2. Reporter Plasmid Design and Cloning
2.3. Peptide Synthesis
2.4. Cu-Catalyzed Azide–Alkyne Cycloaddition for Intein-Activated CPP NF55 Synthesis
2.5. Transfection and Fusion Protein Production
2.6. Flow Cytometry
2.7. Confocal Microscopy
3. Results
3.1. Design of Nanoentity for Intracellular Protein Delivery
3.2. Split EGFP Assay for Bioconjugation Method Validation in Mammalian Cells
3.3. Synthesis of the Nanoentity Substrate Compounds
3.3.1. Substrate 1: Biosynthesis of EGFP–InteinN Fusion Protein
3.3.2. Substrate 2: Chemical Synthesis of Inteinc-activated NF55 Peptide
- (1)
- NF55, with extended C-terminus to include propargylglycine for later click reaction, was synthesized and labeled with carboxytetramethylrhodamine (Rhod).
- (2)
- InteinC peptide was designed to contain azide group.
- (3)
- The fluorolabeled NF55 and azide–inteinC peptide were then conjugated through copper-catalyzed azide–alkyne click reaction. The successful formation of the nanoentity substrate was confirmed via UPLC (Figure S3).
3.4. Assembly of the full Nanoentity and Protein Transduction in Mammalian Cells
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Name | Advantages | Limitations |
---|---|---|
Click chemistry [32,33] | Highly efficient, specific, and bio-orthogonal | Requires specific reactive groups and is not universally suitable for all biomolecules |
Sortase-mediated ligation [32] | Site-specific, efficient, and allows for the incorporation of synthetic peptides into proteins | Limited to those containing a specific motif, potentially requires optimization for various substrates, and has limited activity and stability |
Native chemical ligation [34,35,36] | Compatible with a broad range of biomolecules and suitable for large proteins | Necessitates a cysteine residue at the ligation site, and reaction rates may be slow for certain substrates |
Protein-trans splicing (NCL analog) [37,38,39,40] | Rapid and can occur in a biological environment without affecting the higher-order structure of the protein | Limited to those containing a specific intein sequence |
Name | Description | Size |
---|---|---|
EGFP | Reporter protein component of the nanoentity. Naturally occurring fluorescent protein derived from the jellyfish Aequorea ictoria. | 714 amino acid residues |
NickFect 55 (NF55) | Cell-penetrating peptide/vehicle of the nanoentity. Analog of CPP Transportan 10. Stearoyl-AGYLLGO*INLKALAALAKAIL-NH2; O*—synthesis continued from the side-chain instead of the alpha-amino group. | 21 amino acid residues |
InteinN | N-terminal split intein fragment. Origin from DNA polymerase III subunit alpha; Anabaena variabilis ATCC 29413. | 80 amino acid residues |
InteinC | C-terminal split intein fragment. Origin from DNA polymerase III subunit alpha; Nostoc sp. ATCC 53789. | 39 amino acid residues |
Name | EGFP Fragment | Spilt Intein Fragment | Control Reporter | |
---|---|---|---|---|
First set | p_egfpN_intN | EGFP N-terminal fragment | inteinN | mCherry (red) |
p_intC_egfpC | EGFP N-terminal fragment | inteinC | SBFP (blue) | |
Second set | p_egfp1-10_intN | EGFP 1–10 domains | inteinN | mCherry (red) |
p_intC_egfp11 | EGFP 11 domain | inteinC | SBFP (blue) |
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Nebogatova, J.; Porosk, L.; Härk, H.H.; Kurrikoff, K. Enhancing Cellular Uptake of Native Proteins through Bio-Orthogonal Conjugation with Chemically Synthesized Cell-Penetrating Peptides. Pharmaceutics 2024, 16, 617. https://doi.org/10.3390/pharmaceutics16050617
Nebogatova J, Porosk L, Härk HH, Kurrikoff K. Enhancing Cellular Uptake of Native Proteins through Bio-Orthogonal Conjugation with Chemically Synthesized Cell-Penetrating Peptides. Pharmaceutics. 2024; 16(5):617. https://doi.org/10.3390/pharmaceutics16050617
Chicago/Turabian StyleNebogatova, Jekaterina, Ly Porosk, Heleri Heike Härk, and Kaido Kurrikoff. 2024. "Enhancing Cellular Uptake of Native Proteins through Bio-Orthogonal Conjugation with Chemically Synthesized Cell-Penetrating Peptides" Pharmaceutics 16, no. 5: 617. https://doi.org/10.3390/pharmaceutics16050617
APA StyleNebogatova, J., Porosk, L., Härk, H. H., & Kurrikoff, K. (2024). Enhancing Cellular Uptake of Native Proteins through Bio-Orthogonal Conjugation with Chemically Synthesized Cell-Penetrating Peptides. Pharmaceutics, 16(5), 617. https://doi.org/10.3390/pharmaceutics16050617