Increased Protein Encapsulation in Polymersomes with Hydrophobic Membrane Anchoring Peptides in a Scalable Process
Abstract
:1. Introduction
2. Results
2.1. Protein Encapsulation in Polymersomes with Hydrophobic Membrane Anchoring Peptides
2.2. Specific Release of Immobilized Protein Domains from the Surface of Polymersomes
3. Discussion
4. Materials and Methods
4.1. Genetic Material and Cloning of Plasmids
4.2. Protein Expression and Purification
4.3. Polymersome Formation and Functionalization
4.4. Dynamic Light Scattering (DLS)
4.5. Size-Exclusion Chromatography (SEC)
4.6. Proteinase K Digest
4.7. Fluorescence and Light Extinction Measurements, Quantification of eGFP and Polymersome Concentrations
4.8. Proteolysis with TEV Protease
4.9. Intein Splicing
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Sample | z-Average, nm | PDI, - | Protein Concentration, g/L | # eGFP per Polymersome on the Outer Surface |
---|---|---|---|---|
No protein | 209.8 ± 1.7 | 0.224 ± 0.017 | 0.00 | - |
eGFP | 217.2 ± 0.9 | 0.241 ± 0.004 | 0.50 | - |
eGFP-L’ | 246.7 ± 7.2 | 0.392 ± 0.018 | 0.49 | 158.4 |
eGFP-Cytb5′ | 251.2 ± 2.4 | 0.395 ± 0.049 | 0.50 | 114.3 |
eGFP-Vam3p’ | 237.2 ± 3.5 | 0.391 ± 0.016 | 0.50 | 44.9 |
eGFP-UBC6′ | 244.8 ± 2.8 | 0.375 ± 0.037 | 0.40 | 78.9 |
Sample | A: Expression Yield, mg/L | B: Relative Fluorescence, % | C: EE%, % | Suitability (A × B × C) × 108 |
---|---|---|---|---|
eGFP-L’ | 5.50 | 103 ± 10 (a) | 5.87 | 33 |
eGFP-Cytb5′ | 3.10 | 103 ± 6 (a) | 6.00 | 19 |
eGFP-Vam3p’ | 3.85 | 27 ± 6 (a) | 25.68 | 27 (b) |
eGFP-UBC6′ | 0.96 | 7 ± 4 | 19.67 | 1.3 (b) |
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Mertz, M.; Castiglione, K. Increased Protein Encapsulation in Polymersomes with Hydrophobic Membrane Anchoring Peptides in a Scalable Process. Int. J. Mol. Sci. 2021, 22, 7134. https://doi.org/10.3390/ijms22137134
Mertz M, Castiglione K. Increased Protein Encapsulation in Polymersomes with Hydrophobic Membrane Anchoring Peptides in a Scalable Process. International Journal of Molecular Sciences. 2021; 22(13):7134. https://doi.org/10.3390/ijms22137134
Chicago/Turabian StyleMertz, Michael, and Kathrin Castiglione. 2021. "Increased Protein Encapsulation in Polymersomes with Hydrophobic Membrane Anchoring Peptides in a Scalable Process" International Journal of Molecular Sciences 22, no. 13: 7134. https://doi.org/10.3390/ijms22137134