Effect of Vitreoscilla hemoglobin on recombinant protein expression and energy and redox state of CHO cells

Keywords: CHO cells; Vitreoscilla hemoglobin; GFP; energy metabolism

Abstract

Vitreoscilla hemoglobin (VHb) expression confers considerable improvements to microbial cell factories. However, the effect of VHb on animal cells has been scarcely studied. To gain insight about the impact of VHb on aerobic energy metabolism and recombinant protein expression, CHO-K1 cells stably expressing the green fluorescent protein (GFP) were obtained and transfected for VHb expression. GFP expression slightly increased, whereas cell viability and the mitochondrial membrane potential were not affected by VHb expression. In contrast, the NAD+/NADH ratio and ATP cell content tended to decrease, while the NADP+/NADPH ratio increased as a consequence of VHb presence. These results suggest that VHb can improve recombinant protein expression in CHO cells by altering the energy metabolism. This can be useful for the design of robust cell factories for industrial applications.

References

Becker, M., Junghans, L., Teleki, A., Bechmann, J., Takors, R. (2019). The less the better: How suppressed base addition boosts production of monoclonal antibodies with Chinese Hamster Ovary cells. Frontiers in Bioengineering and Biotechnology, 7, 76.

Chong, W.P.K, Reddy S.G., Yusufi, F.N.K., Lee, D.Y., Wong N.S.C., Heng, C.G., Yap, M. G., Ho, Y.S. (2010). Metabolomics-driven approach for the improvement of chinese hasmster ovary cell growth: overexpression of malate dehydrogenase II. Journal of Biotechnology, 147, 116-121.

De La Vega, J., Braak, B.T., Azzoni, A.R., Monteiro, G.A., Prazeres, D.M.F. (2013). Impact of plasmid quality on lipoplex-mediated transfection. Journal of Pharmaceutical Sciences 102, 3932-3941.

Fischer, S., Handrick, R., Otte, K. (2015). The art of CHO cell engineering: A comprehensive retrospect and future perspectives. Biotechnology Advances 33, 1878-1896.

Ghorbaniagdam, A., Chen, J. Henry, O., Jolicoeur, M. (2014). Analyzing clonal variation of monoclonal antibody-producing CHO cell lines using an In Silico metabolomics platform. PLoS One 9, e90832.

Gorman, C., Arope, S., Grandjean, M., Girod, P.A., Mermod, N. (2009). Use of MAR elements to increase the production of recombinant proteins. In: Al-Rubeai, M. (Ed.), Cell Line Development, Cell Engineering, vol 6. Springer, Dordrecht, pp. 1-32.

Gupta, S.K. Srivastava, S.K., Sharma, A., Nalage, V.H.H., Salvi, D., Kushwaha, H., Chitnis, N.B., Shukla, P. (2017). Metabolic engineering of CHO cells for the development of a robust protein production platform. PLoS One, 12, e0181455.

Harcum, S.W., Lee, K.H. (2016). CHO Cells can make more protein. Cell Systems, 3, 412-413.

Hosios, A.M. Vander Heiden, M.G. (2018). The redox requirements of proliferating mammalian cells. Journal of Biological Chemical, 293, 7490–7498.

Jayasinghe, S. N. (2020). Reimagining Flow Cytometric Cell Sorting. Advanced Biosystems, 4, 1-11.

Jaén, K.E., Velázquez, D., Delvigne, F., Sigala, J.C., Lara, A.R. (2019). Engineering E. coli for improved microaerobic pDNA production. Bioprocess and Biosystems Engineering, 42, 1457-1466.

Juárez, M., González-De la Rosa, C.H., Memún, E., Sigala, J.C., Lara, A.R. (2017). Aerobic expression of Vitreoscilla hemoglobin improves the growth performance of CHO-K1 cells. Biotechnology Journal, 12, 1600438.

Misaghi, S., Shaw, D., Louie, S., Nava, A., Simmons, L., Snedecor, B. (2016). Slashing the timelines: opting to generate high-titer clonal lines faster via viability-based single cell sorting. Biotechnology Progress.32, 198-207.

Pendse, G.J., Bailey, J.E. (1994). Effect of Vitreoscilla hemoglobin expression on growth and specific tissue plasminogen activator productivity in recombinant Chinese hamster ovary cells. Biotechnology and Bioengineering, 44, 1367–1370.

Perry, S.W., Norman, J.P., Barbieri, J., Brown, E.B., Gelbard, H.A. (2011). Mitochondrial membrane potential probes and the proton gradient: a practical usage guide. Biotechniques, 50, 98-115.

Pfizenmaier, J., Matuszczyk, J.C., Takors, R. (2015). Changes in intracellular ATP-content of CHO cells as response to hyperosmolality. Biotechnology Progress, 31, 1212-1216.

Pfizenmaier, J., Junghans, L., Teleki, A., Takors, R. (2016). Hyperosmotic stimulus study discloses benefits in ATP supply and reveals miRNA/mRNA targets to improve recombinant protein production of CHO cells. Biotechnology Journal, 11, 1037–1047.

Reinhart, D., Damjanovik, L., Kaisermayer, C., Sommeregger, W., Gili, A., Gasselhuber, B., Castan, A., Mayrhofer, P., Grünwald-Gruber, C., Kunert, R. (2019). Bioprocessing of recombinant CHO-K1, CHO-DG44, and CHO-S: CHO expression hosts favor either mAb production or biomass synthesis. Biotechnology Journal, 14, 1-11.

Stark, B.C., Dikshit, K.L., Pagilla, K. (2012). The biochemistry of Vitreoscilla hemoglobin. Computational and Structural Biotechnology Journal, 3, 1-8.

Suthar, D.H., Chattoo, B.B. (2006). Expression of Vitreoscilla hemoglobin enhances growth and levels of α-amylase in Schwanniomyces occidentalis. Applied Microbiology and Biotechnology, 72, 94-102.

Tsai, P.S., Hatzimanikatis, V., Bailey, J.E. (1995). Effect of Vitreoscilla hemoglobin dosage on microaerobic Escherichia coli carbon and energy metabolism. Biotechnology and Bioengineering, 49, 139-150.

Walsh, G. (2018). Biopharmaceuticals benchmarks. Nature Biotechnology, 36, 1136-1145.

Wilkens, C.A., Gerdtzen, Z.P. (2015). Comparative metabolic analysis of CHO cell clones obtained through cell engineering, for IgG productivity, growth and cell longevity. PLOS One, 10, 1-15.

Wang, X., Sun, Y., Shen, X., Ke, F., Zhao, H., Liu, Y., Xu, L., Yan, Y. (2012). Intracellular expression of Vitreoscilla hemoglobin improves production of Yarrowia lipolytica lipase LIP2 in a recombinant Pichia pastoris. Enzyme and Microbial Techology, 50, 22-28.

Wu, J.M., Fu, W.C. (2012). Intracellular co-expression of Vitreoscilla hemoglobin enhances cell performance and β-galactosidase production in Pichia Pastoris. Journal of Bioscience and Bioengineering, 113, 332-337.

Wurm, F.M., Wurm, M.J. (2017). Cloning of CHO Cells, productivity and genetic stability—a discussion. Processes, 5, 20.

Zhang, L., Li, Y., Wang, Z., Xia, Y., Chen, W., Tang, K. (2007). Recent developments and future prospects of Vitreoscilla hemoglobin application in metabolic engineering, Biotechnology Advances, 25, 123-136.

Published
2020-10-06
How to Cite
Juárez, M., González de la Rosa, C., Sigala Alanis, J., & Lara-Rodríguez, A. (2020). Effect of Vitreoscilla hemoglobin on recombinant protein expression and energy and redox state of CHO cells. Revista Mexicana De Ingeniería Química, 20(1), 281-288. https://doi.org/10.24275/rmiq/Bio1866
Section
Biotechnology