Interleukin-25 (IL-17E) is a novel Th2 pro-inflammatory cytokine belongs to the member of IL-17 cytokine family. In the present study, bioactive recombinant human IL-25 (rhIL-25), the cDNA of mature IL-25 was synthesized using nested PCR and codon bias of prokaryotic host Escherichia coli. The desired template was cloned into the MCS region of expression vector pET28 a+. The recombinant vector was transformed into maintenance host Escherichia coli DH5α and the transformants were selected by kanamycin resistance marker. Expression was carried out using IPTG inducible Escherichia coli BL21(DE3) in different media like LB, terrific broth and M9 media. Among all, terrific broth was used for the enhanced production of rhIL-25. SDS-PAGE analysis shows 31 kDa proteins against low molecular weight protein marker. Refolding of inclusion bodies with denaturation buffer (25 mM Tris-HCl [pH 7.2], 5 M Urea, 20 mM β-ME and 200 mM NaCl) yields the rhIL-25 at a concentration of ~ 86 mg/L at 37 0 C, where it is high when compared with the expression at 20 0 C (~ 16.5 mg/L). Western blot analysis was carried out using anti human IL-17E/IL-25 antibodies. Biological activity of rhIL-25 was determined by the release of IL-6 from PBMC cells. For the first time, under the conditions of current good manufacturing practice (cGMP), bioactive recombinant IL-25 was produced at large scale in soluble form using industrially feasible bacterial host Escherichia coli BL21(DE3).
Interleukin-17E (also known as is the distinct member of Interleukin-17 cytokine family, induces the expression of . Economical production of interleukin -17E has lot of importance in the current day research in several clinical applications. The objective of the study was to optimize the physico-chemical parameters i.e., dissolved oxygen (DO) and nutritional factors i.e., carbon, nitrogen and phosphate sources on production of Interleukin-17E using industrially important salt inducible Escherichia coli GJ1158. The expression levels were not increased beyond 30 % DO in batch fermentation, but expression levels were increased beyond 30 % DO in fed-batch fermentation. The threshold level of dissolved oxygen ranges was 50 % in respect to the IL-17E production. Pulses of nutritional factors i.e., glucose, yeast extract and K 2 HPO 4 enhanced the expression levels in fed batch fermentation at 40 % and 50 % dissolved oxygen ranges. When OD 600 of the culture reaches to 74 in fed batch fermentation, culture was induced with 100 mM sterile NaCl and further incubated for next 15 hr. Purification was carried out using Ni -NTA spin column. A final concentration of 98 mg/L of purified IL-17E was obtained using cost effective medium viz., modified M9ON medium. The IL-17E thus produced is tested for its activity. In this study, fed-batch fermentation emphasizes the highest concentration of codon optimized recombinant human interleukin-17E using salt inducible expression host till to date, which manages to satisfy the industrial and clinical requirements.
The newly discovered Th2 pro-inflammatory cytokine, interleukin-17E belongs to the member of IL-17 family. In this study, bioactive recombinant mutated human
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