Soluble expression of human recombinant interleukin 11 in SHuffle T7 E. coli using pET system
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zahra miri,1 Mahboobeh Forouzanfar,2 kianoush dormiani,3,*
1. Department of Biology, Faculty of Science and Technology, ACECR Institute of Higher Education, Isfahan, Iran
2. Department of Animal Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
3. Department of Animal Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
- Introduction: Human interleukin-11 (hIL-11) is a cytokine that has multiple physiological properties involving hematologic, immunomodulatory, and epithelial effects. Recombinant hIL-11 has been shown to increase the platelet counts in animals and human. It is the only drug approved for use in chemotherapy-induced thrombocytopenia (CIT). The recombinant form of the cytokine differs from the native protein by of a proline residue at the amino terminus, resulting in a 177-amino acid protein. Oprelvekin is the drug form of recombinant hIL-11 that stimulates megakaryocytopoiesis and thrombopoiesis. In studies of mice and nonhuman primate with moderate and severe myelosuppression or compromised hematopoiesis, oprelvekin was shown to potently induce thrombopoiesis, improve platelet nadirs and accelerate platelet recoveries compared to the control groups. The drug has other important effects such as regulates intestinal epithelium growth, inhibiting adipogenesis, suppressing the pro-inflammatory cytokines release by macrophages, and inducing acute-phase protein synthesis. In the present study, the coding sequence of hIL-11 with His-tag in its upstream was inserted into pET15b/Trx that contained an independent cassette for expression of thioredoxin. The aim of the construction of recombinant plasmid (pET15b/Trx/hIL-11) was to simultaneously but separately express of recombinant hIL-11 along with thioredoxin as a solubilizing tag. The co-expression of thioredoxin was accomplished to improve the production of the soluble and active form of recombinant hIL-11 in SHuffle T7 E. coli as the host cell with enhanced capacity to correctly fold protein with multiple disulfide bonds. Meanwhile, with separate co-expression of hIL-11 with thioredoxin, it is no need to digest the expressed protein and isolate the solubilizing tag from the recombinant cytokine.
- Methods: In this study, the CDS of human IL-11 with a His-tag in the structure of pGH plasmid was digested with appropriate restriction enzymes and was sub-cloned into pET15b contained the a separate expression cassette of thioredoxin (pET15b/Trx/hIL-11). Finally, the plasmid was transformed into SHuffle bacteria, which were cultured in LB broth medium containing ampicillin. The positive colonies that contained pET15b/Trx/hIL-11 were selected. Successful expression of the soluble recombinant hIL-11 protein in bacterial cells was achieved using IPTG induction when the OD600 was approximately 0.6.
- Results: Induction of recombinant protein production was performed using 0.1 mM IPTG at 30 °C for 6 h. After this time period, the cells were harvested and lysed with cell lytic solution. Total protein was extracted and the expression of the recombinant protein was evaluated using 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Eventually, the recombinant hIL-11 protein was purified with Ni-affinity resin and analyzed again with 15% SDS-PAGE.
- Conclusion: In the present study, a new subspecies of Shuffle E. coli was obtained by transformation of an expression plasmid, pET15b/Trx/hIL-11, which was efficiently produced the soluble recombinant hIL-11. Production of the soluble recombinant cytokine was verified through SDS-PAGE analysis. Moreover, the efficient purification of recombinant hIL-11 with Ni-affinity agarose was confirmed by SDS-PAGE in which a protein band was observed with the size of about 24 kDa in comparison to the negative control contained a sample of the extracted protein from the E. coli cells harboring intact pET15b plasmid.
- Keywords: Human Interleukin 11,Recombinant protein, Soluble expression , pET system
