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).

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Production of antimicrobial peptides has gained lot of significance in the present day research. Most of the recombinant proteins are generally produced from IPTG inducible E. coli BL21(DE3). As an alternative, considering the factors like cost and toxic nature of IPTG, salt inducible Escherichia coli GJ1158 was used in the present study for the production of synthetic cationic antimicrobial peptide by fed batch fermentation. This study was conducted to optimize the physico-chemical parameters viz., dissolved oxygen concentration (DOC) and nutritional factors viz., carbon, nitrogen and phosphate sources on bacterial growth and peptide production. Even after increase in DOC more than 30 % in batch culture has no effect on expression, but significant improvement in fed-batch cultivation was observed beyond 30% DOC. Supplementation of production medium with different pulses of nutrient sources like dextrose mono hydrate, yeast extract and Na 2 HPO 4 enhanced the expression in fed batch fermentation process even without disturbing the cell growth at 40 % DOC. When growth reached 15 g/L of dry cell weight, culture was induced with 150 mM NaCl and further cultured for next 15 hr (16.37 g/L dry cell weight). Approximately, 258 mg/L of pure peptide was obtained by using modified GYEON medium. The peptide thus produced is tested for its antimicrobial activity, devoid of hemolytic activity. The fed-batch fermentation which emphasizes, this was the highest reported concentration of recombinant synthetic peptide from salt inducible expression host till to date, which manages to gratify the present day industrial production of the peptides cost-effectively.

Objective: Synthetic cationic antimicrobial peptide (SC-AMP) is an important and upcoming therapeutic molecule against conventional antibiotics. In this study, an attempt was made to purify the SC-AMP without the enzymatic cleavage of the affinity tag, by using an intein-based system. Methods:The intein sequence was amplified from pTYB11 vector using PCR methodologies and the N-terminal of intein was ligated with SC-AMP. The designed construct, intein-SC-AMP was cloned into MCS region of cold shock expression vector, pCOLDI and the recombinant peptide was purified on a chitin affinity column by cleaving intein with 50 mM DTT without applying enzymatic cleavage. Later the peptide was quantified and its antibacterial activity of the purified peptide was studied using well diffusion method.Results: Initially, intein-SC-AMP was expressed as a fusion protein in both IPTG inducible E. coli BL21(DE3) and salt inducible E. coli GJ1158. Single step purification using CBD (chitin binding domain) -intein tag in salt inducible E. coli GJ1158, yields the SC-AMP in the soluble form at a concentration of 208 mg/L. The antibacterial activity and minimal inhibitory concentration (MIC) of the purified SC-AMP was studied against both Gram positive and Gram negative microorganisms. Conclusion:For the first time, single step purification of soluble SC-AMP was carried out using chitin-binding domain affinity tag in salt inducible E. coli GJ1158 without an application of enzymatic cleavage.

Engineered synthetic cationic antimicrobial peptides are the potential alternative drugs to existing antibiotics. In the present study, a novel attempt for the intracellular production of engineered synthetic cationic antimicrobial peptide (escAMP) using Pichia pastoris was studied. The engineered synthetic cationic antimicrobial peptide gene was synthesized using overlapping PCR. An entirokinase and hydroxylamine hydrochloride cleavage sites are incorporated at N-and C-terminal end of escAMP respectively for easy purification. Later the gene was inserted into the MCS region of pPICZ-B vector. The synthetic peptide under the AOX1 promoter was integrated into the Pichia pastoris GS115 genome and the recombinant clones were screened by using antibiotic resistance. Expression profiles of recombinant peptide were done using glycerol and methanol based synthetic medium and analysed on 18 % Tricine-SDS-PAGE. Purification of the expressed peptide was done after cell disruption (10 cycles on time, 10 cycles off time and 10 min of total time) using 6X histidine tag followed by enzymatic cleavage. In this study, 67 gm of dry cell weight/L and 580 mg/L of purified escAMP was produced. The purified peptide is analysed for its anti microbial activity against different Gram positive and Gram negative microbes. For the first time smallest engineered synthetic cationic peptide was designed, cloned and expressed from methanol inducible Pichia pastoris GS115 and production ranges are encouraging.