Productions of solubilized human recombinant interleukin 11 SHuffle T7 strain
نویسندگان: Zahra Miri, Forouzanfar M, Dormiani K ©
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Background and Aim
Human interleukin 11 (hIL-11) is a pleiotropic cytokine secreted by bone marrow stromal cells or mesenchymal cells. hIL-11 plays important roles in numerous biological regulatory processes such as the hematopoietic system, and is anti-inflammatory and anti-apoptotic. The rhIL-11 has been shown to increase platelet counts in animals and humans and is the only drug approved for application in chemotherapy-induced thrombocytopenia (CIT). Oprelvekin, the active ingredient in Neumega®, is a rhIL-11 product, which is produced in Escherichia coli (E. coli) by recombinant DNA technology. With a molecular mass of approximately 19,000 daltons for the non-glycosylated rhIL-11, the protein is composed of 177 amino acids in length in comparison to the natural hIL-11 with 178 amino acids. However, the recombinant form displays a biological activity similar to the natural form both in vitro and in vivo. Oprelvekin as a drug works by stimulating megakaryocytopoiesis and thrombopoiesis. In mice and nonhuman primate studies of animals with moderate and severe myelosuppression, in addition to compromised hematopoiesis, oprelvekin was shown to potently induce thrombopoiesis and improve the platelet nadirs, and accelerated platelet recoveries compared to the controls. In animal studies, oprelvekin was also shown to regulate intestinal epithelium growth by enhancing healing of gastrointestinal lesions.
In this study, the CDS of human IL-11 with a His-tag in the structure of pGH plasmid was digested with suitable restriction enzymes and was sub-cloned into pET15b contained the expression cassette of thioredoxin (pET15b/Trx/hIL-11). Finally, the plasmid was transformed into SHuffle bacteria, which cultured in LB medium containing ampicillin. The positive colonies that contained pET15b/Trx/hIL-11 was selected. Successful expression of the soluble rhIL-11 protein in LB medium was achieved by IPTG induction when the OD600 was approximately 0.8.
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 the expression of the recombinant protein was evaluated using 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Eventually, rhIL-11 protein was purified with Ni-affinity resin, and analyzed again with 15% SDS-PAGE.
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 rhIL-11. Production of soluble recombinant hIL-11 was verified through SDS-PAGE analysis. Moreover, the efficient purification of rhIL-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 vector.
Human interleukin 11 (hIL-11); E. coli; recombinant vector
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