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Production of Recombinant Human Factor VIII in Different Cell Lines and the Effect of Human XBP1 Co-Expression

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Abstract

Recombinant factor VIII is one of the most complex mammalian proteins and a biotechnology venture required for the treatment of hemophilia A. The complexity of the protein, post-translational modifications and limitations of expression elements make the production of active recombinant FVIII a challenge. Here we report the production of biologically active Factor VIII in two different cell lines, CHO and HepG2, by transient transfection. Two expression vectors based on the CMV promoter were used: one harboring CMV Intron A (InA) and the other without it. To bypass difficulties in secretion, we also studied the influence of co-expression of the human splice isoform of the XBP1 gene. We report the production of recombinant FVIII possessing bioengineered FVIII heavy and light chains, linked by a minimal B domain. In our study, HepG2, a human hepatocyte cell line, expressed Factor VIII ten-fold more than a CHO cell line, and in HepG2 cells, the expression of XBP1 improved Factor VIII activity. For CHO cells, expression was improved by the presence of InA, but no further improvement was noted with XBP1 co-expression. These data suggest that the minimal B domain rFVIII preserves Factor VIII biological activity and that different expression elements can be used to improve its production.

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References

  1. Kaufman, R. J., & Pipe, S. W. (1999). Regulation of factor VIII expression and activity by von Willebrand factor. Thrombosis and Haemostasis, 82, 201–208.

    PubMed  CAS  Google Scholar 

  2. Lenting, P. J., van Mourik, J. A., & Mertens, K. (1998). The life cycle of coagulation factor VIII in view of its structure and function. Blood, 92, 3983–3996.

    PubMed  CAS  Google Scholar 

  3. Li, X., & Gabriel, D. A. (1997). The physical exchange of factor VIII (FVIII) between von Willebrand factor and activated platelets and the effect of the FVIII Bdomain on platelet binding. Biochemistry, 36, 10760–10767.

    Article  PubMed  CAS  Google Scholar 

  4. Courter, S. G., & Bedrosian, C. L. (2001). Clinical evaluation of B-domain deleted recombinant factor VIII in previously treated patients. Seminars in Hematology, 38, 44–51.

    Article  PubMed  CAS  Google Scholar 

  5. Dorner, A. J., Bole, D. G., & Kaufman, R. J. (1987). The relationship of N-linked glycosylation and heavy chainbinding protein association with the secretion of glycoproteins. Journal of Cell Biology, 105, 2665–2674.

    Article  PubMed  CAS  Google Scholar 

  6. Dorner, A. J., Wasley, L. C., & Kaufman, R. J. (1989). Increased synthesis of secreted proteins induces expression of glucose-regulated proteins in butyratetreated Chinese hamster ovary cells. Journal of Biological Chemistry, 264, 20602–20607.

    PubMed  CAS  Google Scholar 

  7. Kaufman, R. J., Wasley, L. C., Davies, M. V., Wise, R. J., Israel, D. I., & Dorner, A. J. (1989). Effect of von Willebrand factor coexpression on the synthesis and secretion of factor VIII in Chinese hamster ovary cells. Molecular and Cellular Biology, 9, 1233–1242.

    PubMed  CAS  Google Scholar 

  8. Dorner, A. J., Wasley, L. C., & Kaufman, R. J. (1992). Overexpression of GRP78 mitigates stress induction of glucose regulated proteins and blocks secretion of selective proteins in Chinese hamster ovary cells. EMBO Journal, 11, 1563–1571.

    PubMed  CAS  Google Scholar 

  9. Lynch, C. M., Israel, D. I., Kaufman, R. J., & Miller, A. D. (1993). Sequences in the coding region of clotting factor VIII act as dominant inhibitors of RNA accumulation and protein production. Human Gene Therapy, 4, 259–272.

    Article  PubMed  CAS  Google Scholar 

  10. Miao, H. Z., Sirachainan, N., Palmer, L., Kucab, P., Cunningham, M. A., Kaufman, R. J., & Pipe, S. W. (2004). Bioengineering of coagulation factor VIII for improved secretion. Blood, 103, 3412–3419.

    Article  PubMed  CAS  Google Scholar 

  11. Toole, J. J., Pittman, D. D., Orr, E. C., Murtha, P., Wasley, L. C., & Kaufman, R. J. (1986). A large region (approximately equal to 95 kDa) of human factor VIII is dispensable for in vitro procoagulant activity. Proceedings of the National Academy of Sciences of the United States of America, 83, 5939–5942.

    Article  PubMed  CAS  Google Scholar 

  12. Pittman, D. D., Alderman, E. M., Tomkinson, K. N., Wang, J. H., Giles, A. R., & Kaufman, R. J. (1993). Biochemical, immunological, and in vivo functional characterization of B-domain-deleted factor VIII. Blood, 81(11), 2925–2935.

    PubMed  CAS  Google Scholar 

  13. Cerullo, V., Seiler, M. P., Mane, V., Cela, R., Kaufman, R. J., Clarke, C., Pipe, S. W., & Lee, B. (2007). Correction of murine Hemophilia A and immunological differences of factor VIII variants delivered by helper-dependent adenoviral vectors. Molecular Therapy. Epub ahead of print.

  14. Tigges, M., & Fussenegger, M. (2006). Xbp1-based engineering of secretory capacity enhances the productivity of Chinese hamster ovary cells. Metabolic Engineering, 8, 264–272.

    Article  CAS  Google Scholar 

  15. Gruss, P., Lai, C. J., Dhar, R., & Khoury, G. (1979). Splicing is a requirement for biogenesis of functional 16S mRNA of simian virus 40. Proceedings of the National Academy of Sciences of the United States of America, 76(9), 4317–4321.

    Article  PubMed  CAS  Google Scholar 

  16. Buchman, A. R., & Berg, P. (1988). Comparison of intron-dependent and intron-independent gene expression. Molecular and Cellular Biology, 8, 4395–4405.

    PubMed  CAS  Google Scholar 

  17. Chang, A. H., Stephan, M. T., & Sadelain, M. (2006). Stem cell-derived erythroid cells mediate long-term systemic protein delivery. Nature Biotechnology, 24(8), 1017–1021.

    Article  PubMed  CAS  Google Scholar 

  18. Xia, W., Bringmann, P., McClary, J., Jones, P. P., Manzana, W., Zhu, Y., Wang, S., Liu, Y., Harvey, S., Madlansacay, M. R., McLean, K., Rosser, M. P., MacRobbie, J., Olsen, C. L., & Cobb, R. R. (2006). High levels of protein expression using different mammalian CMV promoters in several cell lines. Protein Expression and Purification, 45(1), 115–124.

    Article  PubMed  CAS  Google Scholar 

  19. Chapman, B. S., Thayer, R. M., Vincent, K. A., & Haigwood, N. L. (1991). Effect of intron A from human cytomegalovirus (Towne) immediate-early gene of heterologous expression in mammalian cells. Nucleic Acids Research, 19(14), 3979–3986.

    Article  PubMed  CAS  Google Scholar 

  20. Xu, Z. L., Mizuguchi, H., Watabe, A. I., Uchida, E., Mayumi, T., & Hayakawa, T. (2001). Optimization of transcriptional regulatory elements for constructing plasmid vectors. Gene, 272, 149–156.

    Article  PubMed  CAS  Google Scholar 

  21. Ruggiero, L. (2002). Clonagem de Anticorpos Recombinantes e Expressão Em Cultura de Células de Ovário Hamister Chinês (CHO), MSc dissertation, Universidade de Brasilia, Brasilia, DF, Brazil.

  22. Herlitschka, S. E., Schlokat, U., Falkner, F. G., & Dorner, F. (1998). High expression of a B-domain deleted factor VIII gene in a human hepatic cell line. Journal of Biotechnology, 61(3), 165–173.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported by grants of FINEP. Campos-da-Paz, M., Costa, C.S., and Simões, I.C. were supported by CNPq/FINEP. The authors thank Msc Izabel Cristina Rodrigues da Silva for assistances with statistical analysis.

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Authors and Affiliations

  1. Departamento de Biologia Celular-IB, Laboratório de Biologia Molecular, Universidade de Brasília, 70910-900, Brasilia, DF, Brazil

    Mariana Campos-da-Paz, Luana Salgado Quilici, Isabella de Carmo Simões, Andrea Queiroz Maranhão & Marcelo Macedo Brigido

  2. Agencia Nacional de Vigilância Sanitária, Ministério da Saúde, Brasilia, Brazil

    Christiane Silva Costa

  3. Laboratório de Microbiologia, Universidade de Brasília, 70910-900, Brasilia, DF, Brazil

    Cynthia Maria Kyaw

Authors
  1. Mariana Campos-da-Paz
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  2. Christiane Silva Costa
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  3. Luana Salgado Quilici
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  4. Isabella de Carmo Simões
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  5. Cynthia Maria Kyaw
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  6. Andrea Queiroz Maranhão
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  7. Marcelo Macedo Brigido
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Correspondence to Marcelo Macedo Brigido.

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Campos-da-Paz, M., Costa, C.S., Quilici, L.S. et al. Production of Recombinant Human Factor VIII in Different Cell Lines and the Effect of Human XBP1 Co-Expression. Mol Biotechnol 39, 155–158 (2008). https://doi.org/10.1007/s12033-008-9055-6

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  • DOI: https://doi.org/10.1007/s12033-008-9055-6

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