Biochemical and Functional Characterization of Mouse Mammary Tumor Virus Full-Length Pr77Gag Expressed in Prokaryotic and Eukaryotic Cells
Abstract
:1. Introduction
2. Materials and Methods
2.1. Nucleotide Numbers
2.2. Full-Length Recombinant Gag Prokaryotic Expression Plasmids
2.3. Full-Length Recombinant Gag Eukaryotic Expression Plasmids
2.4. Escherichia coli Strains and Growth Media
2.5. Expression of Recombinant Full-Length Pr77Gag-His6-Tagged Protein in Bacteria
2.6. Affinity Purification and Gel Filtration Chromatography
2.7. Expression of Recombinant Full-Length Pr77Gag-His6-Tagged Protein in Eukaryotic Cells
2.8. Estimation of RNA Packaging Potential by Reverse Transcriptase PCR
2.9. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Western Blotting
2.10. Detection of Prokaryotically-Expressed Virus-Like Particles Using Transmission Electron Microscopy
2.11. In Vitro Assembly of Virus-Like Particles from Bacterially-Expressed Recombinant Full-Length Pr77Gag-His6-Tag Protein
3. Results and Discussion
3.1. Successful Expression of Full-Length Recombinant MMTV Pr77Gag-His6-Tagged Protein in Bacteria
3.2. Full-Length MMTV Pr77Gag-His6-Tagged Fusion Protein Is Expressed in the Soluble Form in Bacteria
3.3. Immobilized Metal Affinity Chromatography Purification of the Soluble Fraction Containing Recombinant Full-Length Pr77Gag-His6-Tagged Fusion Protein
3.4. Gel Filtration Chromatography Purification of the IMAC-Purified Recombinant Full-Length Pr77Gag-His6-Tagged Fusion Protein
3.5. In Vitro Assembly to Form Virus-Like Particles by the Recombinant Full-Length Pr77Gag-His6-Tagged Fusion Protein
3.6. Recombinant Full-Length MMTV Pr77Gag-His6-Tagged Protein Expressed in Bacteria Can Form Virus-Like Particles
3.7. Eukaryotically-Expressed, Full-Length Recombinant Pr77Gag His6-Tagged Fusion Protein Can Form Virus-Like Particles Competent to Package Unspliced Sub-Genomic RNA
4. Conclusions
Supplementary Materials
Supplementary File 1Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Chameettachal, A.; Pillai, V.N.; Ali, L.M.; Pitchai, F.N.N.; Ardah, M.T.; Mustafa, F.; Marquet, R.; Rizvi, T.A. Biochemical and Functional Characterization of Mouse Mammary Tumor Virus Full-Length Pr77Gag Expressed in Prokaryotic and Eukaryotic Cells. Viruses 2018, 10, 334. https://doi.org/10.3390/v10060334
Chameettachal A, Pillai VN, Ali LM, Pitchai FNN, Ardah MT, Mustafa F, Marquet R, Rizvi TA. Biochemical and Functional Characterization of Mouse Mammary Tumor Virus Full-Length Pr77Gag Expressed in Prokaryotic and Eukaryotic Cells. Viruses. 2018; 10(6):334. https://doi.org/10.3390/v10060334
Chicago/Turabian StyleChameettachal, Akhil, Vineeta Narayana Pillai, Lizna Mohamed Ali, Fathima Nuzra Nagoor Pitchai, Mustafa Taleb Ardah, Farah Mustafa, Roland Marquet, and Tahir Aziz Rizvi. 2018. "Biochemical and Functional Characterization of Mouse Mammary Tumor Virus Full-Length Pr77Gag Expressed in Prokaryotic and Eukaryotic Cells" Viruses 10, no. 6: 334. https://doi.org/10.3390/v10060334
APA StyleChameettachal, A., Pillai, V. N., Ali, L. M., Pitchai, F. N. N., Ardah, M. T., Mustafa, F., Marquet, R., & Rizvi, T. A. (2018). Biochemical and Functional Characterization of Mouse Mammary Tumor Virus Full-Length Pr77Gag Expressed in Prokaryotic and Eukaryotic Cells. Viruses, 10(6), 334. https://doi.org/10.3390/v10060334