Article Search
닫기

Microbiology and Biotechnology Letters

보문(Article)

View PDF

Microbial Biotechnology (MB)  |  Protein Structure, Function, and Engineering

Microbiol. Biotechnol. Lett. 2021; 49(1): 75-87

https://doi.org/10.48022/mbl.2009.09008

Received: September 16, 2020; Accepted: December 24, 2020

Heterologous Expression of Interferon α-2b in Lactococcus lactis and its Biological Activity against Colorectal Cancer Cells

Lita Meilina1,2, Sri Budiarti 2,3, Apon Zaenal Mustopa1*, Huda Shalahudin Darusman4, Lita Triratna1, Muhammad Ajietuta Nugraha5, Muhammad Sabiq Bilhaq6, and Ratih Asmana Ningrum1

1Research Center for Biotechnology, Indonesian Institute of Science (LIPI), Bogor, Indonesia 2School of Biotechnology, IPB University, Bogor, Indonesia 3Indonesia Research Center for Bioresources and Biotechnology, IPB University, Bogor, Indonesia 4Primate Study Center, IPB University, Bogor, Indonesia 5Departement of Biochemistry, Faculty of Mathematics and Natural Science, IPB University, Bogor, Indonesia 6Faculty of Technobiology, Sumbawa University of Technology, Sumbawa, Indonesia

Correspondence to :
Apon Zaenal Mustopa,
azmustopa@yahoo.com

Type I Interferons (IFNα) are known for their role as biological anticancer agents owing to their cell-apoptosis inducing properties. Development of an appropriate, cost-effective host expression system is crucial for meeting the increasing demand for proteins. Therefore, this study aims to develop codon-optimized IFNα- 2b in L. lactis NZ3900. These cells express extracellular protein using the NICE system and Usp45 signal peptide. To validate the mature form of the expressed protein, the recombinant IFNα-2b was screened in a human colorectal cancer cell line using the cytotoxicity assay. The IFNα-2b was successfully cloned into the pNZ8148 vector, thereby generating recombinant L. lactis pNZ8148-SPUsp45-IFNα-2b. The computational analysis of codon-optimized IFNα-2b revealed no mutation and amino acid changes; additionally, the codon-optimized IFNα-2b showed 100% similarity with native human IFNα-2b, in the BLAST analysis. The partial size exclusion chromatography (SEC) of extracellular protein yielded a 19 kDa protein, which was further confirmed by its positive binding to anti-IFNα-2b in the western blot analysis. The crude protein and SEC-purified partial fraction showed IC50 values of 33.22 μg/ml and 127.2 μg/ml, respectively, which indicated better activity than the metabolites of L. lactis NZ3900 (231.8 μg/ml). These values were also comparable with those of the regular anticancer drug tamoxifen (105.5 μg/ml). These results demonstrated L. lactis as a promising host system that functions by utilizing the pNZ8148 NICE system. Meanwhile, codonoptimized usage of the inserted gene increased the optimal protein expression levels, which could be beneficial for its large-scale production. Taken together, the recombinant L. lactis IFNα-2b is a potential alternative treatment for colorectal cancer. Furthermore, its activity was analyzed in the WiDr cell line, to assess its colorectal anticancer activities in vivo.

Keywords: Colorectal cancer, pNZ8148, IFN&alpha,-2b, recombinant protein, Lactococcus lactis, WiDr

  1. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. 2010. Estimates of worldwide burden of cancer in 2008. Int. J. Cancer. 127: 2893-2917.
    Pubmed
  2. World Health Organization. 2019. Indonesia source GLOBOCAN 2018. Int. Agency Res. Cancer. 256: 1-2.
  3. Elsaleh H, Grieu F, Joseph DJ, Iacopetta B. 2001. Molecular epidemiology of colorectal cancer. Int. J. Radiat. Oncol. 51: 260-261.
  4. Pardini B, Kumar R, Naccarati A, Novotny J, Prasad RB, Forsti A, et al. 2011. 5-Fluorouracil-based chemotherapy for colorectal cancer and MTHFR/MTRR genotypes. Br. J. Clin. Pharmacol. 72:162-163.
    Pubmed KoreaMed
  5. Wells JM, Mercenier A. 2008. Mucosal delivery of therapeutic and prophylactic molecules using lactic acid bacteria. Nature 6: 349362.
    Pubmed KoreaMed
  6. Ningrum RA. 2014. Human Interferon Alpha-2b: A Therapeutic Protein for cancer treatment. Scientifica 2014: 1-9.
    Pubmed KoreaMed
  7. Ningrum RA, Wisnuwardhani PH, Santoso A, Herawati N. 2015. Antiproliferative activity of recombinant human interferon alpha2b on estrogen positive human breast cancer MCF-7 cell line. Indones J. Pharm. 26: 86.
  8. Eguchi H, Nagano H, Yamamoto H, Miyamoto A, Kondo M, Dono K, et al. 2000. Augmentation of antitumor activity of 5-fluorouracil by interferon α is associated with up-regulation of p27Kip1 in human hepatocellular carcinoma cells 1. Clin. Cancer Res. 6: 28812890.
  9. Badan Pengawasan Obat dan Makanan Republik Indonesia. 2005. Peraturan Badan pengawas Obat dan Makanan Republik Indonesia. Badan Pengawas Obat. dan. Makanan. 53: 1689-1699.
  10. Kementerian Kesehatan Republik Indonesia. 2015. Rencana Strategis Kementerian Kesehatan Tahun 2015-2019, Kepmenkes RI Nomor HK.02.02/Menkes/52/2015. Kemenkes RI. 248.
  11. Ningrum RA. 2017. Human interferon Alpha2a as anti Hepatitis B and C. Indones. J. Clin. Pharm. 6: 298-310.
  12. Gull I, Samra ZQ, Aslam MS, Athar MA. 2013. Heterologous expression, immunochemical and computational analysis of recombinant human interferon alpha 2b. Springerplus 2: 264.
    Pubmed KoreaMed
  13. Noureddin M, Ghany MG. 2010. Pharmacokinetics and pharmacodynamics of peginterferon and ribavirin: implications for clinical efficacy in the treatment of chronic Hepatitis C. Gastroenterol Clin. North Am. 39: 649-6580.
    Pubmed KoreaMed
  14. Zhao HL, Xue C, Du JL, Ren M, Xia S, Liu ZM. 2012. Balancing the pharmacokinetics and pharmacodynamics of interferon-α2b and human serum albumin fusion protein by proteolytic or reductive cleavage increases its in vivo therapeutic efficacy. Mol. Pharm. 9: 664-6702.
    Pubmed
  15. Ningrum RA, Wardhani WK, Wahyuni I, Mustopa AZ. 2018. Optimization of expression condition, two dimensional and melting point-based characterization of recombinant human interferon alpha-2a fusion and non fusion forms. Ann. Bog. 22: 57.
  16. Ningrum RA, Santoso A, Herawati N. 2017. Overproduction, purification and characterization of human interferon alpha2ahuman serum albumin fusion protein produced in methilotropic yeast Pichia pastoris. J. Phys. Conf. Ser. 835: 1.
  17. Mustopa AZ, Wijaya SK, Ningrum RA, Agustiyanti DF, Triratna L, Alfisyahrin WN. 2019. The expression of codon optimised hepatitis B core antigen (HBcAg) of subgenotype B3 open reading frame in Lactococcus lactis. Microbiol. Biotechnol. Lett. 47: 449458.
  18. Zhoua Z, Danga Y, Zhou M, Li L, Yu CH, Fu J, et al. 2016. Codon usage is an important determinant of gene expression levels largely through its effects on transcription. Proc. Natl. Acad. Sci. USA 113: E6117-E6125.
    Pubmed KoreaMed
  19. Mustopa AZ, Mariyah S, Fatimah, Budiarti S, Murtiyaningsih H, Alfisyahrin WN. 2018. Construction, heterologous expression, partial purification, and in vitro cytotoxicity of the recombinant plantaricin E produced by Lactococcus lactis against Enteropathogenic Escherichia coli K.1.1 and human cervical carcinoma (HeLa) cells. Mol. Biol. Rep. 45: 1235-12448.
    Pubmed
  20. Anwar RI, Mustopa AZ, Ningrum RA. 2019. Construction and expression of indonesian hepatitis B core antigen (HBcAg) in Lactococcus lactis as potential therapeutic vaccine. Biotechnologia 100: 37-45.
  21. Sambrook J, Green MR. 2012. Molecular Cloning: A Laboratory Manual. pp. 1616. 4th Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  22. Borenfreund E, Babich H, Martin-Alguacil N. 1988. Comparisons of two in vitro cytotoxicity assays--the neutral red (nr) and tetrazolium mtt tests. Toxic. in Vitro. 2. 1: 1-6.
  23. Bolotin A, Wincker P, Mauger S, Jaillon O, Malarme K, Weissenbach J, et al. 2001. The complete genome sequence of the lactic acid bacterium. Genome Res. 11: 731-753.
    Pubmed KoreaMed
  24. Kumaran J, Wei L, Kotra LP, Fish EN. 2007. A structural basis for interferon-α-receptor interactions. FASEB J. 21: 3288-32967.
    Pubmed
  25. MoBiTec. 2015. NICE. Expression System for Lactococcus lactis. Available from http://www.mobitec.com/cms/download/Handbooks/NICE_Expression_System-Handbook.pdf. Accessed Aug.8, 2019.
  26. Berlec A, Štrukelj B. 2009. Large increase in brazzein expression achieved by changing the plasmid/strain combination of the NICE system in Lactococcus lactis. Lett. Appl. Microbiol. 48: 750755.
    Pubmed
  27. Borrero J, Jiménez JJ, Gútiez L, Herranz C, Cintas LM, Hernández PE. 2011. Use of the usp45 lactococcal secretion signal sequence to drive the secretion and functional expression of enterococcal bacteriocins in Lactococcus lactis. Appl. Microbiol. Biotechnol. 89:131-143.
    Pubmed
  28. Aitken A, Learmonth MP. 2002. Protein determination by UV absorption, the protein protocols handbook. pp. 3-6. 2nd Ed. Totowa, New Jersey.
  29. Ruiz L, Aroche K, Reyes N. 2006. Aggregation of recombinant human interferon alpha 2b in solution: Technical note. AAPS Pharm. Sci. Technol. 7: 5-9.
    Pubmed KoreaMed
  30. Viola R, Nyvall P, Pelloux J, Davies HV, Pederse M. 1999. Purification and characterisation of a novel starch synthase selective for uridine 5'-diphosphate glucose from the red alga Gracilaria tenuistipitata. Planta. 185: 143-152.
    Pubmed
  31. Winikoff SE, Zeh HJ, Demarco R, Lotze MT. 2006. Cytolytic Assays. pp. 827-849. Pittsburgh, USA.
  32. Meerloo J van, Kaspers GJ, Cloos J. 2011. Cancer cell culture, MTT assay. Methods Mol. Biol. 731: 79-91.
  33. Katla S, Karmakar B, Tadi SRR, Mohan N, Anand B, Pal U, et al. 2019. High level extracellular production of recombinant human interferon alpha 2b in glycoengineered Pichia pastoris: culture medium optimization, high cell density cultivation and biological characterization. J. Appl. Microbiol. 126: 1438-1453.
    Pubmed
  34. Ekowati H, Astuti I, Mustofa M. 2010. Anticancer activity of calanone on HeLa cell line. Indones J. Chem. 10: 240-244.
  35. Suganya M, Gnanamangai BM, Ravindran B, Chang SW, Selvaraj A , Govindasamy C, et al. 2019. Antitumor effect of proanthocyanidin induced apoptosis in human colorectal cancer (HT-29) cells and its molecular docking studies. BMC Chem. 13: 1-14.
    Pubmed KoreaMed
  36. Bazhanova ED. 2005. Participation of interferon-alpha in regulation of apoptosis. J. Evol. Biochem. Physiol. 41: 127-133.
  37. Kementerian Kesehatan Republik Indonesia. 2018. Pedoman Nasional Pelayanan Kedokteran Tata Laksana Kanker Kolorektal:Kepmenkes RI Nomor HK.01.07/MENKES/406/2018. Kemenkes RI.
  38. Jonasch E. 2001. Interferon in oncological practice: review of interferon biology, clinical applications, and toxicities. Oncologist 6: 34-55.
    Pubmed
  39. Wang ZH, Wang YL, Zeng XY. 2014. Construction and expression of a heterologous protein in Lactococcus lactis by using the nisincontrolled gene expression system: The case of the PRRSV ORF6 gene. Genet. Mol. Res. 13: 1088-1096.
    Pubmed
  40. Abbas HT, Kylä-Nikkilä K, Ra R, Saris PEJ. 2006. Nisin induction without nisin secretion. Microbiology 152: 1489-1496.
    Pubmed
  41. Mustopa AZ, Murtiyaningsih H, Fatimah, Suharsono. 2016. Cloning and heterologous expression of extracellular Plantaricin F produced by Lactobacillus plantarum S34 isolated from ‘Bekasam’ in Lactococcus lactis. Microbiol. Indones. 10: 95-106.
  42. Lages AC, Mustopa AZ, Sukmarini L, Suharsono. 2015. Cloning and expression of Plantaricin W produced by Lactobacillus plantarum U10 isolate from ‘Tempoyak’ Indonesian fermented food as immunity protein in Lactococcus lactis. Appl. Biochem. Biotechnol. 177: 909-922.
    Pubmed
  43. Loir YL, Azevedo V, Oliveira SC, Freitas DA, Miyoshi A, BermúdezHumarán LG, et al. 2005. Protein secretion in Lactococcus lactis:An efficient way to increase the overall heterologous protein production. Microb. Cell Fact. 4: 1-13.
  44. Bermúdez-Humarán LG, Gilbert S, Langella P, Commissaire J, Loir YL, L’Haridonr R, et al. 2003. Controlled intra- or extracellular production of staphylococcal nuclease and ovine omega interferon in Lactococcus lactis. FEMS Microbiol. Lett. 224: 307-313.
  45. Bayat O, Baradaran A, Ariff A, Mohamad R, Rahim RA. 2014. Intracellular production of IFN-alpha 2b in Lactococcus lactis. Biotechnol. Lett. 36: 581-585.
    Pubmed
  46. Zhuang Z, Wu Z, Chen M, Wang PG. 2008. Secretion of human interferon-β 1b by recombinant Lactococcus lactis. Biotechnol. Lett. 30: 1819-1823.
    Pubmed
  47. Zhang Q, Zhong J, Liang X, Liu W, Huan L. 2010. Improvement of human interferon alpha secretion by Lactococcus lactis. Biotechnol. Lett. 32: 1271-1277.
    Pubmed
  48. Zhang Q, Zhong J, Huan L. 2011. Expression of hepatitis B virus surface antigen determinants in Lactococcus lactis for oral vaccination. Microbiol. Res. 166: 111-120.
    Pubmed
  49. Riss TL, Moravec RA, Niles AL, Duellman S, Benink HA, Worzella TJ, et al. 2016. Cell viability assays, pp. 295-305. Minor L, Lemmon V, Napper A, Peltier JM, Nelson H, Gal-Edd N (eds.), Assay Guidance Manual, Eli Lilly & Company and the National Center for Advancing Translational Sciences, Bethesda (MD).
  50. Wang W, Roberts CJ. 201. Protein aggregation-Mechanisms, detection, and control. Int. J. Pharm. 550: 251-268.
    Pubmed

Starts of Metrics

Share this article on :

  • mail

Related articles in MBL

Most KeyWord ?

What is Most Keyword?

  • It is most registrated keyword in articles at this journal during for 2 years.