Enhancing the Expression and Purification of IL-29: A study of autoinduction and one-step Purification Methods
Abstract
Background: Cytokines have long been viewed as a sign of hope due to their immunomodulatory and therapeutic characteristics. Developing simple, economical and readily scaled technologies to simplify their manufacturing is a critical challenge.
Method: In this study we have used a customized medium to automatically induce the expression of the IL-29 in E. coli expression system from the T7 promoter, allowing for higher yields as compared to the traditional technique of IPTG induction. Similarly, one-step purification method is employed to make the fermentation process cost-effective, along with enhancing its efficiency.
Results: From 1 L batches of IPTG-induced and autoinduced media, the harvested biomass was 11.8 g and 13.4 g, respectively and their corresponding IBs were 3.8 g and 4.8 g. Total protein purified from 1 L batch was 132 mg, at a concentration of 13 mg/mL, with an indicated high purity of 97%. IL-29 significantly decrease the metabolic activity of HepG2 cells. Specifically, 50% of the cells died at a concentration of 0.156 μg/mL, while 80% of the cells died at a concentration of 5 μg/mL.
Conclusion: This study presents an economical solution for producing and purifying IL-29 in E. coli, resulting in higher yields of biomass and IBs than expensive traditional method. The purified protein was highly pure and had immunomodulatory effects on HepG2 cells. These findings have important implications for developing simplified and scalable technologies for cytokine production with therapeutic potential.
Keywords: Escherichia coli; Cytokines; Interleukins; Interferons; Protein purification
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Berraondo P, Sanmamed MF, Ochoa MC, Etxeberria I, Aznar MA, et al. Cytokines in clinical cancer immunotherapy. British journal of cancer, (2019); 120(1): 6-15.
Sleijfer S, Bannink M, Van Gool AR, Kruit WH, Stoter G. Side effects of interferon-a therapy. Pharmacy world and science, (2005); 27423-431.
Andreakos E, Zanoni I, Galani IE. Lambda interferons come to light: dual function cytokines mediating antiviral immunity and damage control. Current opinion in immunology, (2019); 5667-75.
Meager A, Heath A, Dilger P, Zoon K, Wadhwa M, et al. Standardization of human IL-29 (IFN-lambda1): establishment of a World Health Organization international reference reagent for IL-29 (IFN-lambda1). Journal of Interferon & Cytokine Research, (2014); 34(11): 876-884.
Chrysanthopoulou A, Kambas K, Stakos D, Mitroulis I, Mitsios A, et al. Interferon lambda1/IL-29 and inorganic polyphosphate are novel regulators of neutrophil-driven thromboinflammation. The Journal of Pathology, (2017); 243(1): 111-122.
Steen HC, Gamero AM. Interferon-lambda as a potential therapeutic agent in cancer treatment. Journal of Interferon & Cytokine Research, (2010); 30(8): 597-602.
Kelm NE, Zhu Z, Ding VA, Xiao H, Wakefield MR, et al. The role of IL-29 in immunity and cancer. Critical Reviews in Oncology/Hematology, (2016); 10691-98.
Koch S, Finotto S. Role of interferon-? in allergic asthma. Journal of innate immunity, (2015); 7(3): 224-230.
Wills-Karp M. Interleukin-13 in asthma pathogenesis. Immunological reviews, (2004); 202(1): 175-190.
O’Brien TR, Thomas DL, Jackson SS, Prokunina-Olsson L, Donnelly RP, et al. Weak induction of interferon expression by SARS-CoV-2 supports clinical trials of interferon lambda to treat early COVID-19. Clinical Infectious Diseases, (2020); 71(6): 1410-1412.
Marcello T, Grakoui A, Barba–Spaeth G, Machlin ES, Kotenko SV, et al. Interferons a and ? inhibit hepatitis C virus replication with distinct signal transduction and gene regulation kinetics. Gastroenterology, (2006); 131(6): 1887-1898.
Kanda T, Wu S, Kiyohara T, Nakamoto S, Jiang X, et al. Interleukin-29 suppresses hepatitis A and C viral internal ribosomal entry site-mediated translation. Viral Immunology, (2012); 25(5): 379-386.
Wang J, Oberley-Deegan R, Wang S, Nikrad M, Funk CJ, et al. Differentiated human alveolar type II cells secrete antiviral IL-29 (IFN-?1) in response to influenza A infection. The Journal of Immunology, (2009); 182(3): 1296-1304.
Qian Z, Travanty EA, Oko L, Edeen K, Berglund A, et al. Innate immune response of human alveolar type ii cells infected with severe acute respiratory syndrome–coronavirus. American journal of respiratory cell and molecular biology, (2013); 48(6): 742-748.
Egli A, Santer DM, O’Shea D, Tyrrell DL, Houghton M. The impact of the interferon-lambda family on the innate and adaptive immune response to viral infections. Emerging microbes & infections, (2014); 3(1): 1-12.
Sommereyns C, Paul S, Staeheli P, Michiels T. IFN-lambda (IFN-?) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo. PLoS pathogens, (2008); 4(3): e1000017.
Novotny LA, Evans JG, Su L, Guo H, Meissner EG. Review of lambda interferons in hepatitis B virus infection: outcomes and therapeutic strategies. Viruses, (2021); 13(6): 1090.
Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli: advances and challenges. Frontiers in microbiology, (2014); 5172.
Kamionka M. Engineering of therapeutic proteins production in Escherichia coli. Current pharmaceutical biotechnology, (2011); 12(2): 268-274.
Studier FW, Moffatt BA. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. Journal of molecular biology, (1986); 189(1): 113-130.
Selas Castiñeiras T, Williams SG, Hitchcock AG, Smith DC. E. coli strain engineering for the production of advanced biopharmaceutical products. FEMS microbiology letters, (2018); 365(15): fny162.
Jia B, Jeon CO. High-throughput recombinant protein expression in Escherichia coli: current status and future perspectives. Open biology, (2016); 6(8): 160196.
Bashir H, Ahmed N, Khan MA, Zafar AU, Tahir S, et al. Evaluating the autoinduction expression system and one-step purification for high-level expression and purification of gallbladder-derived rhIL-1Ra. Biotechnology and Applied Biochemistry, (2017); 64(1): 20-26.
Studier FW. Protein production by auto-induction in high-density shaking cultures. Protein expression and purification, (2005); 41(1): 207-234.
Gupta V, Sengupta M, Prakash J, Tripathy BC, Gupta V, et al. Production of recombinant pharmaceutical proteins. Basic and applied aspects of biotechnology, (2017); 77-101.
Schumann W, Ferreira LCS. Production of recombinant proteins in Escherichia coli. Genetics and Molecular Biology, (2004); 27442-453.
DOI: http://dx.doi.org/10.62940/als.v10i1.1730
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