Comparison of Human Factor IX Abundance and mRNA Expression Levels In Stable Insect and Mammalian Cells

Document Type : Research Articles

Authors

• Jafar Vatandoost ¹
• Shadi Tirdad ²

¹ Hakim Sabzevari University

² Islamic Azad University

https://doi.org/10.22067/jcmr.v9i1.61046

Abstract

In the production of recombinant proteins, the selection of an expression system is very important. Although CHO cells are used as mammalian expression system, the ability of insect Schneider line 2 (S2) cells as a new expression system for the high production of many human proteins were confirmed. It is suggested that high copy number of an introduced gene and so high transcription in these cells can be regarded as one of the reasons for high expression of recombinant proteins. Therefore, the present study aimed to evaluate the correlation of recombinant human coagulation factor IX (hFIX) abundance and mRNA expression. The amount of hFIX mRNA by quantitative real-time PCR as well as hFIX protein in cell lysate and cultured media by Elisa was analyzed. The results of data analysis indicated 6 fold increases in mRNA level in S2 cells in comparison to CHO cells. Furthermore, S2 cell line indicated 5.5 and 7-fold increase in total and secreted protein level, respectively, compared to CHO cell line. The data demonstrated the correlation of mRNA and protein abundance and indicate that S2 cell lines are superior in producing the recombinant proteins.

Keywords

• Drosophila S2 cells
• CHO cells
• Coagulation factor IX
• Real-Time PCR
• ELISA

20.1001.1.20089147.2017.9.1.2.7

References

1.Bernard A., Kost T., Overton L., Cavegn C., Young J., Bertrand M., Yahia-Cherif Z., Chabert C. and Mills A. (1994) Recombinant protein expression in a Drosophila cell line: comparison with the baculovirus system. In Cell Culture Engineering IV. Springer. 139-144.

2.Brown T. A. (2016) Gene cloning and DNA analysis: an introduction. John Wiley & Sons.

3.Cherbas L. and Cherbas P. (2007) Transformation of Drosophila cell lines: an alternative approach to exogenous protein expression. Baculovirus and Insect Cell Expression Protocols:317-340.

4.Haddad‐Mashadrizeh A., Zomorodipour A., Izadpanah M., Sam M. R., Ataei F., Sabouni F. and Hosseini S. J. (2009) A systematic study of the function of the human β‐globin introns on the expression of the human coagulation factor IX in cultured Chinese hamster ovary cells. The Journal of Gene Medicine 11:941-950.

5.Johansen H., van der Straten A., Sweet R., Otto E., Maroni G. and Rosenberg M. (1989) Regulated expression at high copy number allows production of a growth-inhibitory oncogene product in Drosophila Schneider cells. Genes & Development 3:882-889.

6.Kim K. R., Kim Y. K. and Cha H. J. (2008) Recombinant baculovirus-based multiple protein expression platform for Drosophila S2 cell culture. Journal of Biotechnology 133:116-122.

7.Kirkpatrick R. B. and Shatzman A. (1999) Drosophila S2 System for Heterologous Gene Expression-11.

8.Maroni G., Otto E. and Lastowski-Perry D. (1986) Molecular and cytogenetic characterization of a metallothionein gene of Drosophila. Genetics 112:493-504.

9.Moraes A. M., Jorge S. A., Astray R. M., Suazo C. A., Riquelme C. E. C., Augusto E. F., Tonso A., Pamboukian M. M., Piccoli R. A. and Barral M. F. (2012) Drosophila Melanogaster S2 cells for expression of heterologous genes: From gene cloning to bioprocess development. Biotechnology Advances 30:613-628.

10.Myhre S., Lingjærde O.-C., Hennessy B. T., Aure M. R., Carey M. S., Alsner J., Tramm T., Overgaard J., Mills G. B. and Børresen-Dale A.-L. (2013) Influence of DNA copy number and mRNA levels on the expression of breast cancer related proteins. Molecular Oncology 7:704-718.

11.Otto E., Allen J., Young J., Palmiter R. and Maroni G. (1987) A DNA segment controlling metal-regulated expression of the Drosophila melanogaster metallothionein gene Mtn. Molecular and Cellular Biology 7:1710-1715.

12.Vatandoost J. and Pakdaman S. F. (2016) The Effects of Influencing Factors on γ-carboxylation and Expression of Recombinant Vitamin K Dependent Coagulation Factors. Journal of Biomedicine 1.

13.Vatandoost J., Zomorodipour A., Sadeghizadeh M., Aliyari R., Bos M. H. and Ataei F. (2012) Expression of biologically active human clotting factor IX in Drosophila S2 cells: γ‐carboxylation of a human vitamin K‐dependent protein by the insect enzyme. Biotechnology Progress 28:45-51.

14.Wikibooks c. (2017) An Introduction to Molecular Biology/Gene Expression. Wikibooks, The Free Textbook Project.