Molecular Cloning, High Level Expression and Activity Analysis of Constructed Human Interleukin–25 Using Industrially Important IPTG Inducible Escherichia coli BL21(DE3)

Abstract

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).

Site Directed Mutagenesis , Molecular Cloning and Expression of interleukin - 17E to Generate Structural Variant with Enhanced Specific Activity Using Industrial Friendly Salt Inducible Escherichia coli GJ1158

“…Further this gene is sequenced (Figure-3) used as template to amplify the structural variant rh17E with a different set of primers in PCR and amplicon is confirmed on 1.2% agarose, followed by sequencing (Figure 4). The results are similar to our previous study [16]. …”

CgIL17-5, an ancient inflammatory cytokine in Crassostrea gigas exhibiting the heterogeneity functions compared with vertebrate interleukin17 molecules

“…In the present study, the content of IL6 in HuVEC cells increased significantly after the treatment of recombinant CgIL17-5. The similar results of IL6 secretion was also reported in stromal cells when they were incubated with vertebrate IL17A, IL17D, IL17E or IL17F (Jaya Lakshmi et al, 2014;Zrioual et al, 2008), rather than IL17B and IL17C (Li et al, 2000), suggesting that vertebrate IL17 family members might differ in modulatory patterns of inflammatory response. It was further confirmed that CgIL17-5 was a primitive IL-17 members, and it could conservatively initiate signal transduction to promote IL6 synthesis as well as inflammatory response, but its precise activating mechanism should be further investigated.…”

Immobilization of Arylmalonate Decarboxylase

“…The enzyme was prepared according to a routinely used protocol using IPTG (isopropyl β-D-1-thiogalactopyranoside) for induction [23]. The presence of the target protein was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Mini PROTEAN ® Tetra Cell, Bio-Rad, Hercules, CA, USA), observing the band of 26 kDa.…”