Molecular Biotechnology

, Volume 61, Issue 2, pp 134–144 | Cite as

Engineering Chlamydomonas reinhardtii for Expression of Functionally Active Human Interferon-α

  • Yassin El-Ayouty
  • Islam El-Manawy
  • Sherif Nasih
  • Emad Hamdy
  • Rashad KebeishEmail author
Original Paper


Human interferon (IFN) are secreted cytokines that play a major regulatory role in response to various infections. Commercially, IFN-α has been approved to treat many chronic viral diseases as well as a variety of cancers and different types of leukemia. In this study, a binary vector containing human IFN-α2a gene under the regulation of the cauliflower mosaic virus 35S promoter was constructed. IFN-œ2a expression cassette was transferred to Chlamydomonas reinhardtii cells via Agrobacterium-mediated transformation method. Three independent transgenic C. reinhartii lines were generated and reported to produce a biologically active IFN-œ2a. The expressed IFN-œ2a was partially purified and tested for their antitumor and antiviral properties. Cytotoxicity and cell apoptosis assays involving the usage of the recombinant C. reinhardtii IFN-œ2a (Cr. IFN-œ2a) against the growth of Hep-G2 cells (human hepatocellular carcinoma), EAC-induced tumors (Ehrlich Ascites Carcinoma) in mice prove the functionality of the produced IFN-œ2a as an anticancer drug. Moreover, Cr.IFN-œ2a is shown to have significant inhibitory effects on the propagation of the vesicular stomatitis virus (VSV). The overall observed results support the application of C. reinhardtii expression system as a cost effective, eco-friendly, safe, and easy to employ compared to plant, bacterial and animal cell culture systems.


Chlamydomonas reinhardtii Recombinant IFN-α2a Antiviral and anticancer activity 



This work was in part under the supervision of the Applied Scientific Research Center, Herbal and Medicinal plants research group, Taibah University. We are thankful to Dr. Ali Fahmy Head of R&D Sector at VACSERA, Egypt for his valuable help with the antitumor and antiviral assays.

Supplementary material

12033_2018_143_MOESM1_ESM.doc (210 kb)
Supplementary material 1 (DOC 210 KB)


  1. 1.
    Abdel-Hamid, M., El-Daly, M., Molnegren, V., El-Kafrawy, S., Abdel-Latif, S., Esmat, G., Strickland, G. T., Loffredo, C., Albert, J., & Widell, A. (2007). Genetic diversity in hepatitis C virus in Egypt and possible association with hepatocellular carcinoma. Journal of General Virology, 88, 1526–1531.CrossRefGoogle Scholar
  2. 2.
    Frank, C., Mohamed, M. K., Strickland, G. T., Lavanchy, D., Arthur, R. R., Magder, L. S., El Khoby, T., Abdel-Wahab, Y., Anwar, W., and Sallam, I. (2000). The role of parenteral antischistosomal therapy in the spread of hepatitis C virus in Egypt. The Lancet, 355, 887–891.CrossRefGoogle Scholar
  3. 3.
    Shepard, C. W., Finelli, L., & Alter, M. J. (2005). Global epidemiology of hepatitis C virus infection. The Lancet Infectious Diseases, 5, 558–567.CrossRefGoogle Scholar
  4. 4.
    Bosinger, S. E., & Utay, N. S. (2015) Type I interferon: Understanding its role in HIV pathogenesis and therapy. Current HIV/AIDS Reports, 12, 41–53.CrossRefGoogle Scholar
  5. 5.
    Liu, C. J., Chuang, W. L., Lee, C. M., Yu, M. L., Lu, S. N., Wu, S. S., Liao, L. Y., Chen, C. L., Kuo, H. T., & Chao, Y. C. (2009) Peginterferon alfa-2a plus ribavirin for the treatment of dual chronic infection with hepatitis B and C viruses. Gastroenterology, 136, 496–504. e493.CrossRefGoogle Scholar
  6. 6.
    Noël, N., Jacquelin, B., Huot, N., Goujard, C., Lambotte, O., & Müller-Trutwin, M. (2018). Interferon-associated therapies toward HIV control: The back and forth. Cytokine and Growth Factor Reviews, 40, 99–112.CrossRefGoogle Scholar
  7. 7.
    Maeda, S., McCandliss, R., Gross, M., Sloma, A., Familletti, P. C., Tabor, J. M., Evinger, M., Levy, W. P., & Pestka, S. (1980) Construction and identification of bacterial plasmids containing nucleotide sequence for human leukocyte interferon. Proceedings of the National Academy of Sciences, 77, 7010–7013.CrossRefGoogle Scholar
  8. 8.
    Li, H., Liu, Q., Cui, K., Liu, J., Ren, Y., & Shi, D. (2012). Expression of biologically active human interferon alpha 2b in the milk of transgenic mice. Transgenic Research, 22, 169–178.CrossRefGoogle Scholar
  9. 9.
    Razaghi, A., Villacrés, C., Jung, V., Mashkour, N., Butler, M., Owens, L., & Heimann, K. (2017). Improved therapeutic efficacy of mammalian expressed-recombinant interferon gamma against ovarian cancer cells. Experimental Cell Research, 359, 20–29.CrossRefGoogle Scholar
  10. 10.
    Mizukami, T., Komatsu, Y., Hosoi, N., Itoh, S., & Oka, T. (1986). Production of active human interferon-α in E. coli I. Preferential production by lower culture temperature. Biotechnology Letters, 8, 605–610.CrossRefGoogle Scholar
  11. 11.
    Ahangarzadeh, S., Daneshvar, M. H., Rajabi-Memari, H., Galehdari, H., & Alamisaied, K. (2012). Cloning, transformation and expression of human interferon-α2b gene in tobacco plant (Nicotiana tabacum cv. xanthi). Jundishapur Journal of Natural Pharmaceutical Products, 7, 111.CrossRefGoogle Scholar
  12. 12.
    Masumura, T., Morita, S., Miki, Y., Kurita, A., Morita, S., Shirono, H., Koga, J., & Tanaka, K. (2006) Production of biologically active human interferon-α2 in transgenic rice. Plant Biotechnology, 23, 91–97.CrossRefGoogle Scholar
  13. 13.
    Barrera, D. J., & Mayfield, S. P. (2013). High-value recombinant protein production in microalgae. Handbook of Microalgal Culture: Applied Phycology and Biotechnology, 2, 532–544.CrossRefGoogle Scholar
  14. 14.
    Griesbeck, C., & Kirchmayr, A. (2012) Algae: An alternative to the higher plant system in gene farming. In Molecular farming in plants: Recent advances and future prospects (pp. 125–143). New York: Springer.CrossRefGoogle Scholar
  15. 15.
    Rosales-Mendoza, S. (2016). Algae-made cytokines and growth factors, in algae-based biopharmaceuticals (pp. 95–108). New York: Springer.CrossRefGoogle Scholar
  16. 16.
    Griesbeck, C., Kobl, I., & Heitzer, M. (2006). Chlamydomonas reinhardtii. Molecular Biotechnology, 34, 213–223.CrossRefGoogle Scholar
  17. 17.
    Munjal, N., Garzon-Sanabria, A. J., Quinones, K. W., Gregory, J., & Nikolov, Z. L. (2014) Light-induced production of an antibody fragment and malaria vaccine antigen from Chlamydomonas reinhardtii. Processes 2, 625–638.CrossRefGoogle Scholar
  18. 18.
    Shamriz, S., & Ofoghi, H. (2018). Engineering the chloroplast of Chlamydomonas reinhardtii to express the recombinant PfCelTOS-Il2 antigen-adjuvant fusion protein. Journal of Biotechnology, 266, 111–117.CrossRefGoogle Scholar
  19. 19.
    Rosales-Mendoza, S., Paz-Maldonado, L. M. T., & Soria-Guerra, R. E. (2012). Chlamydomonas reinhardtii as a viable platform for the production of recombinant proteins: Current status and perspectives. Plant Cell Reports, 31, 479–494.CrossRefGoogle Scholar
  20. 20.
    Gorman, D. S., & Levine, R. (1965) Cytochrome f and plastocyanin: Their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi. Proceedings of the National Academy of Sciences, 54, 1665–1669.CrossRefGoogle Scholar
  21. 21.
    Antonelli, G. (2008). Biological basis for a proper clinical application of alpha interferons. The New Microbiologica, 31, 305–318.Google Scholar
  22. 22.
    Kebeish, R., Aboelmy, M., El-Naggar, A., El-Ayouty, Y., & Peterhansel, C. (2015). Simultaneous overexpression of cyanidase and formate dehydrogenase in Arabidopsis thaliana chloroplasts enhanced cyanide metabolism and cyanide tolerance. Environmental and Experimental Botany, 110, 19–26.CrossRefGoogle Scholar
  23. 23.
    Koncz, C., & Schell, J. (1986). The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Molecular and General Genetics MGG, 204, 383–396.CrossRefGoogle Scholar
  24. 24.
    Kumar, S. V., Misquitta, R. W., Reddy, V. S., Rao, B. J., & Rajam, M. V. (2004). Genetic transformation of the green alga-”Chlamydomonas reinhardtii by Agrobacterium tumefaciens. Plant Science, 166, 731–738.CrossRefGoogle Scholar
  25. 25.
    Mohkami, A., Marashi, H., Shahriary Ahmadi, F., Tohidfar, M., & Mohsenpour, M. (2015). Evaluation of agrobacterium-mediated transformation of Chlamydomonas reinhardtii using a synthetic amorpha-4, 11-diene synthase gene. Journal of Cell and Molecular Research, 7, 53–58.Google Scholar
  26. 26.
    Chomczynski, P., & Mackey, K. (1995). Substitution of chloroform by bromo-chloropropane in the single-step method of RNA isolation. Analytical Biochemistry, 225, 163–164.CrossRefGoogle Scholar
  27. 27.
    Niessen, M., Thiruveedhi, K., Rosenkranz, R., Kebeish, R., Hirsch, H.-J., Kreuzaler, F., & Peterhänsel, C. (2007). Mitochondrial glycolate oxidation contributes to photorespiration in higher plants. Journal of Experimental Botany, 58, 2709–2715.CrossRefGoogle Scholar
  28. 28.
    Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.CrossRefGoogle Scholar
  29. 29.
    Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal of Immunological Methods, 65, 55–63.CrossRefGoogle Scholar
  30. 30.
    Mishra, S., Tamta, A. K., Sarikhani, M., Desingu, P. A., Kizkekra, S. M., Pandit, A. S., Kumar, S., Khan, D., Raghavan, S. C., & Sundaresan, N. R. (2018). Subcutaneous Ehrlich Ascites Carcinoma mice model for studying cancer-induced cardiomyopathy. Scientific Reports, 8, 5599.CrossRefGoogle Scholar
  31. 31.
    Quinn, R., Kashiwagi, M., Moore, R. E., & Norton, T. R. (1974). Anticancer activity of zoanthids and the associated toxin, palytoxin, against ehrlich ascites tumor and P-388 lymphocytic leukemia in mice. Journal of Pharmaceutical Sciences, 63, 257–260.CrossRefGoogle Scholar
  32. 32.
    Ozgur, O., Karti, S., Sonmez, M., Yilmaz, M., Karti, D., Ozdemir, F., & Ovali, E. (2003). Effects of interferon-alfa-2a on human hepatoma HepG2 cells. Experimental Oncology, 25, 105–107.Google Scholar
  33. 33.
    Bussereau, F., Flamand, A., & Pese-Part, D. (1982). Reproducible plaquing system for rabies virus in CER cells. Journal of Virological Methods, 4, 277–282.CrossRefGoogle Scholar
  34. 34.
    Lansky, E. P., & Newman, R. A. (2007). Punica granatum (pomegranate) and its potential for prevention and treatment of inflammation and cancer. Journal of Ethnopharmacology, 109, 177–206.CrossRefGoogle Scholar
  35. 35.
    Buathong, R., Hermann, L., Thaisomboonsuk, B., Rutvisuttinunt, W., Klungthong, C., Chinnawirotpisan, P., Manasatienkij, W., Nisalak, A., Fernandez, S., & Yoon, I.-K. (2015). Detection of Zika virus infection in Thailand, 2012 and 2014. The American Journal of Tropical Medicine and Hygiene, 93, 380–383.CrossRefGoogle Scholar
  36. 36.
    Barta, A., Sommergruber, K., Thompson, D., Hartmuth, K., Matzke, M. A., & Matzke, A. J. (1986). The expression of a nopaline synthase—human growth hormone chimaeric gene in transformed tobacco and sunflower callus tissue. Plant Molecular Biology, 6, 347–357.CrossRefGoogle Scholar
  37. 37.
    Hiatt, A., Caffferkey, R., & Bowdish, K. (1989). Production of antibodies in transgenic plants. Nature, 342, 76.CrossRefGoogle Scholar
  38. 38.
    Obembe, O. O., Popoola, J. O., Leelavathi, S., & Reddy, S. V. (2011). Advances in plant molecular farming. Biotechnology Advances, 29, 210–222.CrossRefGoogle Scholar
  39. 39.
    Harris, E. H. (2001). Chlamydomonas as a model organism. Annual Review of Plant Biology, 52, 363–406.CrossRefGoogle Scholar
  40. 40.
    Cerutti, H., Johnson, A. M., Gillham, N. W., & Boynton, J. E. (1997). A eubacterial gene conferring spectinomycin resistance on Chlamydomonas reinhardtii: integration into the nuclear genome and gene expression. Genetics, 145, 97–110.Google Scholar
  41. 41.
    Kozminski, K. G., Diener, D. R., & Rosenbaum, J. L. (1993). High level expression of nonacetylatable alpha tubulin in Chlamydomonas reinhardtii. Cell Motility and the Cytoskeleton, 25, 158–170.CrossRefGoogle Scholar
  42. 42.
    Tang, D., Qiao, S.-Y., & Wu, M. (1995). Insertion mutagenesis of Chlamydomonas reinhardtii by electroporation and heterologous DNA. Biochemistry and Molecular Biology International, 36, 1025–1035.Google Scholar
  43. 43.
    Heitzer, M., Eckert, A., Fuhrmann, M., & Griesbeck, C. (2007). Influence of codon bias on the expression of foreign genes in microalgae, in transgenic microalgae as green cell factories (pp. 46–53). New York: Springer.CrossRefGoogle Scholar
  44. 44.
    Elshal, M. F., & McCoy, J. P. (2006) Multiplex bead array assays: Performance evaluation and comparison of sensitivity to ELISA. Methods 38, 317–323.CrossRefGoogle Scholar
  45. 45.
    El-Garhy, F. M., Badr, D. A., & Fahmy, A. (2017). Assessment of anti-cancer and anti-viral potential of pomegranate peel extract against human prostate, and larynx cancer cell lines: In-vitro Study. Cancer Biology, 7, 65–73.Google Scholar
  46. 46.
    Kebeish, R., El-Sayed, A., Fahmy, H., & Abdel-Ghany, A. (2016) Molecular cloning, biochemical characterization, and antitumor properties of a novel L-asparaginase from Synechococcus elongatus PCC6803. Biochemistry (Moscow), 81, 1173–1181.CrossRefGoogle Scholar
  47. 47.
    Elshafei, A. M., Hassan, M. M., Abd, M., Abouzeid, E., Mahmoud, D. A., & Elghonemy, D. H. (2011). Purification, characterization and antitumor activity of L-asparaginase from Penicillium brevicompactum 2 NRC 829 3. British Microbiology Research Journal, 2, 158–174.CrossRefGoogle Scholar
  48. 48.
    Gladilina, Y. A., Sokolov, N., & Krasotkina, J. (2009). Cloning, expression, and purification of Helicobacter pylori L-asparaginase. Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry, 3, 89–91.CrossRefGoogle Scholar
  49. 49.
    Enomoto, H., Tao, L., Eguchi, R., Sato, A., Honda, M., Kaneko, S., Iwata, Y., Nishikawa, H., Imanishi, H., & Iijima, H. (2017). The in vivo antitumor effects of type I-interferon against hepatocellular carcinoma: The suppression of tumor cell growth and angiogenesis. Scientific Reports, 7, 12189.CrossRefGoogle Scholar
  50. 50.
    Melén, K., Keskinen, P. a., Lehtonen, A., & Julkunen, I. (2000). Interferon-induced gene expression and signaling in human hepatoma cell lines. Journal of Hepatology, 33, 764–772.CrossRefGoogle Scholar
  51. 51.
    Zhou, Z., Hamming, O. J., Ank, N., Paludan, S. r. R., Nielsen, A. L., & Hartmann, R. (2007). Type III interferon (IFN) induces a type I IFN-like response in a restricted subset of cells through signaling pathways involving both the Jak-STAT pathway and the mitogen-activated protein kinases. Journal of Virology, 81, 7749–7758.CrossRefGoogle Scholar
  52. 52.
    Hagiwara, S., Kudo, M., Nakatani, T., Sakaguchi, Y., Nagashima, M., Fukuta, N., Kimura, M., Hayakawa, S., & Munakata, H. (2007). Combination therapy with PEG-IFN-α and 5-FU inhibits HepG2 tumour cell growth in nude mice by apoptosis of p53. British Journal of Cancer, 97, 1532.CrossRefGoogle Scholar
  53. 53.
    Paul, F., Pellegrini, S., & Uzé, G. (2015). IFNA2: The prototypic human alpha interferon. Gene, 567, 132–137.CrossRefGoogle Scholar
  54. 54.
    Lai, C. L., Lau, J. Y., Wu, P. C., Ngan, H., Chung, H. T., Mitchell, S. J., Corbett, T. J., Chow, A. W., & Lin, H. J. (1993). Recombinant interferon-alpha in inoperable hepatocellular carcinoma: A randomized controlled trial. Hepatology, 17, 389–394.CrossRefGoogle Scholar
  55. 55.
    Llovet, J. M., Sala, M., Castells, L. s., Suarez, Y., Vilana, R., Bianchi, L. S., Ayuso, C., Vargas, V. c., Rodés, J., & Bruix, J. (2000). Randomized controlled trial of interferon treatment for advanced hepatocellular carcinoma. Hepatology, 31, 54–58.CrossRefGoogle Scholar
  56. 56.
    Kotredes, K. P., & Gamero, A. M. (2013). Interferons as inducers of apoptosis in malignant cells. Journal of Interferon and Cytokine Research, 33, 162–170.CrossRefGoogle Scholar
  57. 57.
    Gamero, A. M., & Larner, A. C. (2001). Vanadate facilitates interferon α-mediated apoptosis that is dependent on the Jak/Stat pathway. Journal of Biological Chemistry, 276, 13547–13553.CrossRefGoogle Scholar
  58. 58.
    Scarzello, A. J., Romero-Weaver, A. L., Maher, S. G., Veenstra, T. D., Zhou, M., Qin, A., Donnelly, R. P., Sheikh, F., & Gamero, A. M. (2007). A Mutation in the SH2 domain of STAT2 prolongs tyrosine phosphorylation of STAT1 and promotes type I IFN-induced apoptosis. Molecular Biology of the Cell, 18, 2455–2462.CrossRefGoogle Scholar
  59. 59.
    Trinchieri, G. (2010). Type I interferon: Friend or foe? Journal of Experimental Medicine, 207, 2053–2063.CrossRefGoogle Scholar
  60. 60.
    Pestka, S., Langer, J. A., Zoon, K. C., & Samuel, C. E. (1987). Interferons and their actions. Annual Review of Biochemistry, 56, 727–777.CrossRefGoogle Scholar
  61. 61.
    Nishiguchi, S., Kuroki, T., Nakatani, S., Morimoto, H., Takeda, T., Nakajima, S., Shiomi, S., Seki, S., Kobayashi, K., & Otani, S. (1995). Randomised trial of effects of interferon-alpha on incidence of hepatocellular carcinoma in chronic active hepatitis C with cirrhosis. The Lancet, 346, 1051–1055.CrossRefGoogle Scholar

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Authors and Affiliations

  1. 1.Botany and Microbiology Department, Faculty of ScienceZagazig UniversityZagazig, SharkiaEgypt
  2. 2.Botany Department, Faculty of ScienceSuez Canal UniveristyIsmailiaEgypt
  3. 3.Clinical Pathology DepartmentKaser El-Aini Medical School - Cairo UniversityCairoEgypt
  4. 4.Biology Department, Faculty of Science YanbuTaibah UniversityYanbu El-BahrSaudi Arabia

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