Cost Effective Production Of Recombinant Human Interleukin24from Escherichia Coli And Anti-cancer Application

3.%)L KH’ J R S LGHQ LILHG0HODQRPD ’LIIH HQ LD LRQ-Associated protein-7(MDA-7)upon discovery of cell surface receptor Mda-7renamed Interleukin24(IL24). IL24has beendesignated as a member of the interleukin10family of cytokines. This family includes IL10,IL19, IL20, IL22, IL24and IL26.IL24has growth suppressive properties in a wide variety ofhuman cancer cell lines without inducing harmful effects in normal cells. IL24has potentialtherapeutic applications and has an important role in tumor cell biology.Recombinant human interleukin24production was initially produced from Escherichiacoli with a conventional method for characterization. The fermentation strategy was based onapplication of LB media for shake flask culture. The high pressure homogenizer was used forcell lyses. The traditional approach for IB solubilization and refolding was applied to producea low sample volume for purification. The anion&cation exchange chromatography wasapplied to remove impurities from the sample and to produce purified product. According toconventional method a negligible rhIL24was produced with more effort and more timeconsuming. A new strategy has been designed for cost effective production of rhIL24on thebasis of conventional method production. The cost and composition of culture media iscritical for commercial-scale production of recombinant proteins in E. coli. Addition of yeastextract and glucose to medium enhances rhIL24production, and the use of lactose instead ofIPTG for induction drops the cost and decreases toxicity. In addition, a two-step denaturingand refolding (2DR) process replaced the conventional method for refolding and improvedrhIL24production. Simple process was developed for quality production of rhIL24with highpurity. LC-MS/MS provides definitive identification and quantitative information on rhIL24.The single step purified rhIL24was further analyzed as potent therapeutic agent forcancer and Multi drug resistance cancer cells. rhIL24displayed biological activity on HepG2hepatocellular carcinoma cells, but no effect on L02cells. We investigated the effect of rhIL-24on the Adriamycin (ADM)-resistant human breast cancer cell line MCF-7/ADM. Thecytotoxicity of rhIL-24and ADM was determined by3-[4,5-dimethylthiazol-2.-yl],5-diphenyl tetrazolium bromide (MTT) assays. The expression of P-gp was assessed byconfocal microscopy and Western blot analysis. The IC50value of Adriamycin in MCF-7/ADM cells decreased in a dose-dependent manner when rhIL-24was used. ADMaccumulation increased while P-gp expression decreased at a low dose (4μPol·L-1) of rhIL24in MCF-7/ADM cells. In vitro biological activity affix the statement that rhIL-24circumvented the drug-resistance of MCF-7/ADM cells via activation of the transcriptionfactor Stat3and rhIl24has potential to act as a P-gp inhibitor to reverse Adriamycinresistance in breast cancer.The final conclusive objective of the current research was formulation of rhIL24ascontrolled release drug. Stem cell-based gene therapies have the limitation of transplantingmodified IL24to subcutaneous tumors. Adenovirus (Ad)-mediated delivery of IL-24cancause growth arrest and apoptosis. Conjugation of the therapeutic compound to polymers hadincreased the aqueous solubility and enhanced the plasma circulation half-life. This work developed a strategy to form Nanoparticles of rhIL24and PEG Protamine complex.PEGylated Protamine-IL24Nanoparticle is able to confirm activity against MCF-7/ADMcells. The current investigation paves the road towards clinical realization of an effectiveformulation for rhIL24controlled release.