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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
Cloning and Expression of IL-15 Gene in E. coli, Rosetta (DE3) Strain
M. Bahrololoumi Shapour Abadi1 , A. Arashkia1 , N. Mohajel1 , F. Roohvand1 , K. Azadmanesh 2
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:   (2428 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.
Keywords: Interleukin-15, Cloning and Expression, Escherichia coli Rosetta (DE3) Strain
Full-Text [PDF 986 kb]   (1137 Downloads)    
Article Type: Original Research | Subject: Biologic Products
Received: 2019/07/1 | Accepted: 2020/05/22
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