Initial Screening of Poly(ethylene glycol) Amino Ligands for Affinity Purification of Plasmid DNA in Aqueous Two-Phase Systems
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Poly(Ethylene Glycol)–Amine Affinity Ligands Synthesis
2.2.1. Methoxypolyethyleneglycol–Lysine and –Arginine
2.2.2. Methoxypolyethyleneglycol–Histidine
2.3. Plasmid Production
2.4. Alkaline Cell Lysis
2.5. Lysate Desalting
2.6. Aqueous Two-Phase Experiments
2.7. Extraction with an Ammonium-Sulphate-Rich Phase
2.8. Nucleic Acids Partitioning Analysis by Agarose Gel Electrophoresis (AGE)
2.9. Total Protein Partitioning Analysis Using Polyacrylamide Denaturing Gel Electrophoresis (SDS–PAGE)
2.10. Total Protein Quantification
2.11. Densitometric Analysis of Agarose Gel Bands
3. Results and Discussion
3.1. Specificity of PEG–Amino Affinity Ligands
3.2. pDNA Extraction with an Ammonium-Sulphate-Rich Phase
3.3. Protein Contamination Assessment
3.4. Process Scale-Up and Capacity Assessment
4. 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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6.5% PEG–Amine | ||||
Top Phase | Bottom Phase | |||
Band Area | Percentage | Band Area | Percentage | |
pDNA | 4525.3 | 5.1 | 58,908.5 | 74.1 |
RNA | 84,927.4 | 94.9 | 20,622.5 | 25.9 |
1.2% PEG–Lysine | ||||
Top Phase | Bottom Phase | |||
Band Area | Percentage | Band Area | Percentage | |
pDNA | 0 | 0 | 50,694.5 | 100 |
RNA | 43254.3 | 100 | 0 | 0 |
0.5% PEG–Arginine | ||||
Top Phase | Bottom Phase | |||
Band Area | Percentage | Band Area | Percentage | |
pDNA | 0 | 0 | 48,787.4 | 100 |
RNA | 54,061.7 | 100 | 0 | 0 |
1.3% PEG–Histidine | ||||
Top Phase | Bottom Phase | |||
Band Area | Percentage | Band Area | Percentage | |
pDNA | 0 | 0 | 47,470.7 | 81.3 |
RNA | 30,013.5 | 100 | 10,904.5 | 18.7 |
0.5% PEG–Arginine (Scale-Up) | ||||
Top Phase | Bottom Phase | |||
Band Area | Percentage | Band Area | Percentage | |
pDNA | 0 | 0 | 29,553.5 | 77.5 |
RNA | 39,394.4 | 100 | 8601.0 | 22.5 |
0.5% PEG–Arginine (High-Yield Cell Lysate) | ||||
Top Phase | Bottom Phase | |||
Band Area | Percentage | Band Area | Percentage | |
pDNA | 0 | 0 | 24,504.4 | 81.8 |
RNA | 28,249.9 | 100 | 5453.5 | 18.2 |
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da Silva, N.R.; Jorge, P.; Martins, J.A.; Teixeira, J.A.; Marcos, J.C. Initial Screening of Poly(ethylene glycol) Amino Ligands for Affinity Purification of Plasmid DNA in Aqueous Two-Phase Systems. Life 2021, 11, 1138. https://doi.org/10.3390/life11111138
da Silva NR, Jorge P, Martins JA, Teixeira JA, Marcos JC. Initial Screening of Poly(ethylene glycol) Amino Ligands for Affinity Purification of Plasmid DNA in Aqueous Two-Phase Systems. Life. 2021; 11(11):1138. https://doi.org/10.3390/life11111138
Chicago/Turabian Styleda Silva, Nuno R., Paula Jorge, José A. Martins, José A. Teixeira, and João C. Marcos. 2021. "Initial Screening of Poly(ethylene glycol) Amino Ligands for Affinity Purification of Plasmid DNA in Aqueous Two-Phase Systems" Life 11, no. 11: 1138. https://doi.org/10.3390/life11111138