Effects of Kifunensine on Production and N-Glycosylation Modification of Butyrylcholinesterase in a Transgenic Rice Cell Culture Bioreactor
Abstract
:1. Introduction
2. Results and Discussion
2.1. Cell Growth Kinetics, Sugar Consumption, and Oxygen Uptake Rate with Kifunensine Addition
2.2. rrBChE Production and Recombinant Protein Purity in the Presence of Kifunensine
2.3. Purification, SDS-PAGE, and Western Blot Analysis of rrBChE with/without Kifunensine Treatment
2.4. Site-Specific N-glycosylation Analysis
3. Materials and Methods
3.1. Transgenic Rice Cell Suspension Inoculum
3.2. Bioreactor Operation with Kifunensine Treatment
3.3. Harvesting of Culture Medium rrBChE and Cell-Associated rrBChE
3.4. Biomass Measurements
3.5. Sugar Analysis
3.6. Active rrBChE and Total Protein Quantification
3.7. Purification of rrBChE
3.8. Gel Electrophoresis and Western Blot
3.9. Site-Specific N-glycopeptide Analysis of rrBChE
4. Conclusions and Future Prospects
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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tgrowth (Days) | x0 (g DW/L) | xf (g DW/L) | µmax (Day−1) | τD (Days) | Max OUR (mmol O2 L−1 h−1) | Max SOUR (mmol O2 g DW−1 h−1) |
---|---|---|---|---|---|---|
7 | 2.53 ± 0.08 | 8.29 ± 0.24 | 0.21 ± 0.02 | 3.3 ± 0.4 | 2.09 ± 0.01 | 0.37 ± 0.01 |
tinduction (Days) | x’0 (g DW/L) | x’f (g DW/L) | g FW/ g DW Ratio† | Max total rrBChE Production (µg/g FW; mg/L) | Max Volumetric Productivity † (µg L−1 Day−1) | Max Specific Productivity † (μg g DW−1 Day−1) |
---|---|---|---|---|---|---|
5 | 10.04 ± 0.53 | 8.24 ± 0.19 | 11.5 ± 0.3 | 79.0 ± 2.2; 7.5 ± 0.4 | 680 ± 34 | 82.5 ± 4.6 |
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Macharoen, K.; Li, Q.; Márquez-Escobar, V.A.; Corbin, J.M.; Lebrilla, C.B.; Nandi, S.; McDonald, K.A. Effects of Kifunensine on Production and N-Glycosylation Modification of Butyrylcholinesterase in a Transgenic Rice Cell Culture Bioreactor. Int. J. Mol. Sci. 2020, 21, 6896. https://doi.org/10.3390/ijms21186896
Macharoen K, Li Q, Márquez-Escobar VA, Corbin JM, Lebrilla CB, Nandi S, McDonald KA. Effects of Kifunensine on Production and N-Glycosylation Modification of Butyrylcholinesterase in a Transgenic Rice Cell Culture Bioreactor. International Journal of Molecular Sciences. 2020; 21(18):6896. https://doi.org/10.3390/ijms21186896
Chicago/Turabian StyleMacharoen, Kantharakorn, Qiongyu Li, Veronica A. Márquez-Escobar, Jasmine M. Corbin, Carlito B. Lebrilla, Somen Nandi, and Karen A. McDonald. 2020. "Effects of Kifunensine on Production and N-Glycosylation Modification of Butyrylcholinesterase in a Transgenic Rice Cell Culture Bioreactor" International Journal of Molecular Sciences 21, no. 18: 6896. https://doi.org/10.3390/ijms21186896