Volume 23, Issue 1 (2020)                   mjms 2020, 23(1): 27-32 | Back to browse issues page

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Bahrololoumi Shapour Abadi M, Arashkia A, Mohajel N, Roohvand F, Azadmanesh K. Cloning and Expression of IL-15 Gene in E. coli, Rosetta (DE3) Strain. mjms 2020; 23 (1) :27-32
URL: http://mjms.modares.ac.ir/article-30-34387-en.html
1- Molecular Virology Department, Pasteur Institute of Iran, Tehran, Iran
2- Molecular Virology Department, Pasteur Institute of Iran, Tehran, Iran , azadmanesh@pasteur.ac.ir
Abstract:   (1818 Views)
Interleukin 15 (IL-15) is a cytokine that, due to its physiological activity, can play a role in anti-cancer therapies. It shows positive effect on Natural Killer cells differentiation, proliferation, activation, and surveillance and also on surveillance of memory CD8+ T cells. However, the expression and purification yield of recombinant IL-15 is low. So, it worth improving the production conditions of this useful protein. Therefore in the present study, cloning and expression of human IL-15 are reported in E. coli, strain Rosetta (DE3) which is different from previous bacterial hosts BL21 strain. This strain (Rosetta DE3) has the potential to use codons rarely used by bacteria and consequently, the expressed protein can be more similar to the human protein. First, the human IL-15 coding sequence was synthetized, and then the sequence was cloned into pET28a plasmid. Confirming the accuracy of the final construct was done by colony PCR, restriction analysis (which was done using BamHI and XhoI restriction enzymes and the expected 391bp band was observed) and sequencing. Then, the recombinant construct was transformed into competent E. coli Rosetta (DE3) bacteria. Expression was done in OD600 of 0.6 in the presence of 1mM Isopropyl ß-D-1-thiogalactopyranoside (IPTG). Protein characterization was done by SDS-PAGE and then by western blotting using a specific commercial anti-IL-15 antibody. The 15kDa band on the gel and blot, showed the presence of IL-15. Densitometry by Fiji software determined 37% production yield. It is expected a suitable function from this produced recombinant cytokine due to expression in E. coli Rosetta (DE3) in future studies.
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Article Type: Original Research | Subject: Biologic Products
Received: 2019/07/1 | Accepted: 2020/05/22

References
1. Grabstein KH, Eisenman J, Shanebeck K, Rauch C, Srinivasan S, Fung V, et al. Cloning of a T cell growth factor that interacts with the beta chain of the interleukin-2 receptor. Science. 1994;264(5161):965-8. [Link] [DOI:10.1126/science.8178155]
2. Carson WE, Giri JG, Lindemann MJ, Linett ML, Ahdieh M, Paxton R, et al. Interleukln (IL) 15 is a novel cytoklne that activates human natural killer cells via components of the IL-2 receptor. J Exp Med. 1994;180(4):1395-1403. [Link] [DOI:10.1084/jem.180.4.1395]
3. Dubois S, Mariner J, Waldmann TA, Tagaya Y. IL-15Rα recycles and presents IL-15 in trans to neighboring cells. Immunity. 2002;17(5):537-47. [Link] [DOI:10.1016/S1074-7613(02)00429-6]
4. Giri JG, Ahdieh M, Eisenman J, Shanebeck K, Grabstein K, Kumaki S, et al. Utilization of the beta and gamma chains of the IL‐2 receptor by the novel cytokine IL‐15. EMBO J. 1994;13(12):2822-30. [Link] [DOI:10.1002/j.1460-2075.1994.tb06576.x]
5. Waldmann TA. The shared and contrasting roles of IL2 and IL15 in the life and death of normal and neoplastic lymphocytes: Implications for cancer therapy. Cancer Immunol Res. 2015;3(3):219-27. [Link] [DOI:10.1158/2326-6066.CIR-15-0009]
6. Fehniger TA, Caligiuri MA. Interleukin 15: Biology and relevance to human disease. Blood. 2001;97(1):14-32. [Link] [DOI:10.1182/blood.V97.1.14]
7. Steel JC, Waldmann TA, Morris JC. Interleukin-15 biology and its therapeutic implications in cancer. Trends Pharmacol Sci. 2012;33(1):35-41. [Link] [DOI:10.1016/j.tips.2011.09.004]
8. Yu P, Steel JC, Zhang M, Morris JC, Waldmann TA. Simultaneous blockade of multiple immune system inhibitory checkpoints enhances antitumor activity mediated by interleukin-15 in a murine metastatic colon carcinoma model. Clin Cancer Res. 2010;16(24):6019-28. [Link] [DOI:10.1158/1078-0432.CCR-10-1966]
9. Zhang M, Yao Z, Dubois S, Ju W, Müller JR, Waldmann TA. Interleukin-15 combined with an anti-CD40 antibody provides enhanced therapeutic efficacy for murine models of colon cancer. Proc Natl Acad Sci. 2009;106(18):7513-8. [Link] [DOI:10.1073/pnas.0902637106]
10. Xu X, Sun Q, Yu X, Zhao L. Rescue of nonlytic Newcastle Disease Virus (NDV) expressing IL-15 for cancer immunotherapy. Virus Res. 2017;233:35-41. [Link] [DOI:10.1016/j.virusres.2017.03.003]
11. Hu XD, Chen ST, Li JY, Yu DH, Cai H. An IL-15 adjuvant enhances the efficacy of a combined DNA vaccine against Brucella by increasing the CD8+ cytotoxic T cell response. Vaccine. 2010;28(12):2408-15. [Link] [DOI:10.1016/j.vaccine.2009.12.076]
12. Wu J. IL-15 agonists: The cancer cure cytokine. J Mol Genet Med. 2013;7:85. [Link]
13. Liu B, Kong L, Han K, Hong H, Marcus WD, Chen X, et al. A novel fusion of ALT-803 (interleukin (IL)-15 superagonist) with an antibody demonstrates antigen-specific antitumor responses. J Biol Chem. 2016;291(46):23869-81. [Link] [DOI:10.1074/jbc.M116.733600]
14. Ward A, Anderson M, Craggs RI, Maltby J, Grahames C, Davies RA, et al. E. coli expression and purification of human and cynomolgus IL-15. Protein Expr Purif. 2009;68(1):42-8. [Link] [DOI:10.1016/j.pep.2009.05.004]
15. Béhar G, Solé V, Defontaine A, Maillasson M, Quéméner A, Jacques Y, et al. Evolution of interleukin-15 for higher E. coli expression and solubility. Protein Eng Des Sel. 2011;24(3):283-90. [Link] [DOI:10.1093/protein/gzq107]
16. Huang H, Luo Y, Baradei H, Liu Sh, Haenssen KK, Sanglikar S, et al. A novel strategy to produce high level and high purity of bioactive IL15 fusion proteins from mammalian cells. Protein Expr Purif. 2018;148:30-9. [Link] [DOI:10.1016/j.pep.2018.03.010]
17. Sun W, Lai Y, Li H, Nie T, Kuang Y, Tang X, et al. High level expression and purification of active recombinant human interleukin-15 in Pichia pastoris. J Immunol Methods. 2016;428:50-7. [Link] [DOI:10.1016/j.jim.2015.12.002]
18. Tegel H, Tourle S, Ottosson J, Persson A. Increased levels of recombinant human proteins with the Escherichia coli strain Rosetta (DE3). Protein Expr Purif. 2010;69(2):159-67. [Link] [DOI:10.1016/j.pep.2009.08.017]
19. Fu W, Lin J, Cen P. 5-Aminolevulinate production with recombinant Escherichia coli using a rare codon optimizer host strain. Appl Microbiol Biotechnol. 2007;75(4):777-82. [Link] [DOI:10.1007/s00253-007-0887-y]
20. Hsu C, Hughes MS, Zheng Z, Bray RB, Rosenberg SA, Morgan RA. Primary human T lymphocytes engineered with a codon-optimized IL-15 gene resist cytokine withdrawal-induced apoptosis and persist long-term in the absence of exogenous cytokine. J Immunol. 2005;175(11):7226-34. [Link] [DOI:10.4049/jimmunol.175.11.7226]
21. Sambrook J. Molecular cloning : A laboratory manual. 3rd Edition. Huntington: Cold Spring Harbor Laboratory; 2003. [Link]
22. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: An open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676-82. [Link] [DOI:10.1038/nmeth.2019]
23. Vyas VV, Esposito D, Sumpter TL, Broadt TL, Hartley J, Knapp IV GC, et al. Clinical manufacturing of recombinant human interleukin 15. I. Production cell line development and protein expression in E. coli with stop codon optimization. Biotechnol Prog. 2012;28(2):497-507. [Link] [DOI:10.1002/btpr.746]

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