Construction and Characterization of Recombinant HEK Cell Over-Expressing TOSO/FAIM3 and Evaluation of its Expression

Document Type : Original Article (s)

Authors

1 Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

2 Associate Professor, Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

3 Assistant Professor, Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

4 PhD Candidate, Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

5 Assistant Professor, Pediatric Inherited Diseases Research Center, Research Institute for Primordial Prevention of Non-communicable Disease AND Department of Genetics and Molecular Biology, School of Medicine, Isfahan University Of Medical Sciences, Isfahan, Iran

Abstract

Background: Integral membrane proteins involve in a variety of valuable cellular processes including signal transduction, transport and cell-to-cell communication. Thus, due to their importance, are targeted to treatment of diseases or disorders. To achieve this goal, it is necessary to obtain adequate membrane proteins. A variety of expression systems have been used to overexpress these proteins but mammalian cells can produce membrane proteins structurally close to natural form of them and are favourable for this purpose. TOSO/FAIM3 (Fas Apoptosis Inhibitory Molecule), highly conserved membrane protein, playing important role in cell survival, immune surveillance, and homeostasis and in this study was overexpressed on HEK-293T membrane.    Methods: Eukaryotic expression vector pEZ-M67-TOSO was transformed into Escherichia coli top 10F’ strain to multiply. Then, the plasmid was extracted and digested with restriction enzyme Eco31I to make a linear plasmid. Subsequently, HEK-293T cells were transfected with linear plasmid and treated with hygromycin to select TOSO expressing stable cells. After three weeks, resistant colons were expanded, Chromosomal DNA and total RNA were extracted and the presence of TOSO-cDNA in HEK-293T genome and the expression level of TOSO were evaluated using polymerase chain reaction (PCR) and Real-time PCR methods, respectively.Findings: The integration of TOSO expressing cassette in HEK-293T genome was confirmed. HEK-293 cells expressed 1700 mRNA TOSO molecule/cell which was in the range of protein with abundant expression.Conclusion: Mammalian cells represent membrane proteins in desirable form versus other expression systems. For this reason, we used HEK293T cells to overexpress transmembrane TOSO protein. In this investigation, we successfully constructed the HEK293 cell line with stable TOSO overexpression, which will facilitate further researches to characterize biophysical and biochemical structure and function of TOSO and pharmaceutical biological researches about it.

Keywords

References

  1. Tan S, Tan HT, Chung MC. Membrane proteins and membrane proteomics. Proteomics 2008; 8(19): 3924-32.
  2. Wallin E, von HG. Genome-wide analysis of integral membrane proteins from eubacterial, archaean, and eukaryotic organisms. Protein Sci 1998; 7(4): 1029-38.
  3. Mao L, Vaiphei ST, Shimazu T, Schneider WM, Tang Y, Mani R, et al. The E. coli single protein production system for production and structural analysis of membrane proteins. J Struct Funct Genomics 2010; 11(1): 81-4.
  4. Andrell J, Tate CG. Overexpression of membrane proteins in mammalian cells for structural studies. Mol Membr Biol 2013; 30(1): 52-63.
  5. Song Y, Jacob CO. The mouse cell surface protein TOSO regulates Fas/Fas ligand-induced apoptosis through its binding to Fas-associated death domain. J Biol Chem 2005; 280(10): 9618-26.
  6. Kubagawa H, Oka S, Kubagawa Y, Torii I, Takayama E, Kang DW, et al. Identity of the elusive IgM Fc receptor (FcmuR) in humans. J Exp Med 2009; 206(12): 2779-93.
  7. Kaveri SV, Silverman GJ, Bayry J. Natural IgM in immune equilibrium and harnessing their therapeutic potential. J Immunol 2012; 188(3): 939-45.
  8. Dharmadhikari G, Muhle M, Schulthess FT, Laue S, Oberholzer J, Pattou F, et al. TOSO promotes beta-cell proliferation and protects from apoptosis. Mol Metab 2012; 1(1-2): 70-8.
  9. Nguyen XH, Lang PA, Lang KS, Adam D, Fattakhova G, Foger N, et al. Toso regulates the balance between apoptotic and nonapoptotic death receptor signaling by facilitating RIP1 ubiquitination. Blood 2011; 118(3): 598-608.
  10. Tate CG, Haase J, Baker C, Boorsma M, Magnani F, Vallis Y, et al. Comparison of seven different heterologous protein expression systems for the production of the serotonin transporter. Biochim Biophys Acta 2003; 1610(1): 141-53.
  11. Chaudhary S, Pak JE, Pedersen BP, Bang LJ, Zhang LB, Ngaw SM, et al. Efficient expression screening of human membrane proteins in transiently transfected Human Embryonic Kidney 293S cells. Methods 2011; 55(4): 273-80.
  12. Wurm FM. Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 2004; 22(11): 1393-8.
  13. Eifler N, Duckely M, Sumanovski LT, Egan TM, Oksche A, Konopka JB, et al. Functional expression of mammalian receptors and membrane channels in different cells. J Struct Biol 2007; 159(2): 179-93.
  14. Prather PL, McGinn TM, Claude PA, Liu-Chen LY, Loh HH, Law PY. Properties of a kappa-opioid receptor expressed in CHO cells: interaction with multiple G-proteins is not specific for any individual G alpha subunit and is similar to that of other opioid receptors. Brain Res Mol Brain Res 1995; 29(2): 336-46.
  15. Chelikani P, Reeves PJ, Rajbhandary UL, Khorana HG. The synthesis and high-level expression of a beta2-adrenergic receptor gene in a tetracycline-inducible stable mammalian cell line. Protein Sci 2006; 15(6): 1433-40.
  16. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: A laboratory manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989.
  17. Vire B, David A, Wiestner A. TOSO, the Fcmicro receptor, is highly expressed on chronic lymphocytic leukemia B cells, internalizes upon IgM binding, shuttles to the lysosome, and is downregulated in response to TLR activation. J Immunol 2011; 187(8): 4040-50.
  18. Brenner D, Brustle A, Lin GH, Lang PA, Duncan GS, Knobbe-Thomsen CB, et al. Toso controls encephalitogenic immune responses by dendritic cells and regulatory T cells. Proc Natl Acad Sci U S A 2014; 111(3): 1060-5.
  19. Bruni R, Kloss B. High-throughput cloning and expression of integral membrane proteins in Escherichia coli. Curr Protoc Protein Sci 2013; 74: Unit.
  20. Parker JL, Newstead S. Method to increase the yield of eukaryotic membrane protein expression in Saccharomyces cerevisiae for structural and functional studies. Protein Sci 2014; 23(9): 1309-14.
  21. Bouvier M, Menard L, Dennis M, Marullo S. Expression and recovery of functional G-protein-coupled receptors using baculovirus expression systems. Curr Opin Biotechnol 1998; 9(5): 522-7.
  22. Ames R, Nuthulaganti P, Fornwald J, Shabon U, van der Keyl H, Elshourbagy N. Heterologous expression of G protein-coupled receptors in U-2 OS osteosarcoma cells. Receptors Channels 2004; 10(3-4): 117-24.
  23. Klammt C, Lohr F, Schafer B, Haase W, Dotsch V, Ruterjans H, et al. High level cell-free expression and specific labeling of integral membrane proteins. Eur J Biochem 2004; 271(3): 568-80.
  24. Sarramegna V, Talmont F, Demange P, Milon A. Heterologous expression of G-protein-coupled receptors: comparison of expression systems from the standpoint of large-scale production and purification. Cell Mol Life Sci 2003; 60(8): 1529-46.
  25. Domingue JC, Ao M, Sarathy J, George A, Alrefai WA, Nelson DJ, et al. HEK-293 cells expressing the cystic fibrosis transmembrane conductance regulator (CFTR): a model for studying regulation of Cl- transport. Physiol Rep 2014; 2(9).
  26. Chaudhary S, Pak JE, Gruswitz F, Sharma V, Stroud RM. Overexpressing human membrane proteins in stably transfected and clonal human embryonic kidney 293S cells. Nat Protoc 2012; 7(3): 453-66.
  27. Wu W, Wang Y, Deng XL, Sun HY, Li GR. Cholesterol down-regulates BK channels stably expressed in HEK 293 cells. PLoS One 2013; 8(11): e79952.
  28. Cudna RE, Dickson AJ. Endoplasmic reticulum signaling as a determinant of recombinant protein expression. Biotechnol Bioeng 2003; 81(1): 56-65.
  29. Knowlton AA. Life, death, the unfolded protein response and apoptosis. Cardiovasc Res 2007; 73(1): 1-2.
  30. Wong DC, Wong KT, Nissom PM, Heng CK, Yap MG. Targeting early apoptotic genes in batch and fed-batch CHO cell cultures. Biotechnol Bioeng 2006; 95(3): 350-61.
  31. Wurtele H, Little KC, Chartrand P. Illegitimate DNA integration in mammalian cells. Gene Ther 2003; 10(21): 1791-9.
Journal of Isfahan Medical School
Volume 33, Issue 324 - Serial Number 324
January and February 2015
Pages 171-182

Files

History

  • Receive Date: 03 March 2015
  • Revise Date: 01 November 2024
  • Accept Date: 06 April 2022

Share

How to cite

Statistics