Browse > Article
http://dx.doi.org/10.48022/mbl.2009.09008

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

Meilina, Lita (Research Center for Biotechnology, Indonesian Institute of Science (LIPI))
Budiarti, Sri (School of Biotechnology, IPB University)
Mustopa, Apon Zaenal (Research Center for Biotechnology, Indonesian Institute of Science (LIPI))
Darusman, Huda Shalahudin (Primate Study Center, IPB University)
Triratna, Lita (Research Center for Biotechnology, Indonesian Institute of Science (LIPI))
Nugraha, Muhammad Ajietuta (Departement of Biochemistry, Faculty of Mathematics and Natural Science, IPB University)
Bilhaq, Muhammad Sabiq (Faculty of Technobiology, Sumbawa University of Technology)
Ningrum, Ratih Asmana (Research Center for Biotechnology, Indonesian Institute of Science (LIPI))
Publication Information
Microbiology and Biotechnology Letters / v.49, no.1, 2021 , pp. 75-87 More about this Journal
Abstract
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 ㎍/ml and 127.2 ㎍/ml, respectively, which indicated better activity than the metabolites of L. lactis NZ3900 (231.8 ㎍/ml). These values were also comparable with those of the regular anticancer drug tamoxifen (105.5 ㎍/ml). These results demonstrated L. lactis as a promising host system that functions by utilizing the pNZ8148 NICE system. Meanwhile, codon-optimized 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; $IFN{\alpha}-2b$; Lactococcus lactis; pNZ8148; recombinant protein; WiDr;
Citations & Related Records
연도 인용수 순위
  • Reference
1 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.   DOI
2 Elsaleh H, Grieu F, Joseph DJ, Iacopetta B. 2001. Molecular epidemiology of colorectal cancer. Int. J. Radiat. Oncol. 51: 260-261.
3 Wells JM, Mercenier A. 2008. Mucosal delivery of therapeutic and prophylactic molecules using lactic acid bacteria. Nature 6: 349-362.   DOI
4 Ningrum RA. 2014. Human Interferon Alpha-2b: A Therapeutic Protein for cancer treatment. Scientifica 2014: 1-9.   DOI
5 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.   DOI
6 Ningrum RA, Santoso A, Herawati N. 2017. Overproduction, purification and characterization of human interferon alpha2a-human serum albumin fusion protein produced in methilotropic yeast Pichia pastoris. J. Phys. Conf. Ser. 835: 1.
7 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.   DOI
8 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.   DOI
9 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: 449-458.   DOI
10 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.
11 Loir YL, Azevedo V, Oliveira SC, Freitas DA, Miyoshi A, BermudezHumaran 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.   DOI
12 Berlec A, Strukelj 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: 750-755.   DOI
13 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.   DOI
14 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.   DOI
15 Sambrook J, Green MR. 2012. Molecular Cloning: A Laboratory Manual. pp. 1616. 4th Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
16 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.   DOI
17 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.
18 Meerloo J van, Kaspers GJ, Cloos J. 2011. Cancer cell culture, MTT assay. Methods Mol. Biol. 731: 79-91.   DOI
19 Aitken A, Learmonth MP. 2002. Protein determination by UV absorption, the protein protocols handbook. pp. 3-6. 2nd Ed. Totowa, New Jersey.
20 Winikoff SE, Zeh HJ, Demarco R, Lotze MT. 2006. Cytolytic Assays. pp. 827-849. Pittsburgh, USA.
21 Ekowati H, Astuti I, Mustofa M. 2010. Anticancer activity of calanone on HeLa cell line. Indones J. Chem. 10: 240-244.   DOI
22 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.   DOI
23 Kementerian Kesehatan Republik Indonesia. 2018. Pedoman Nasional Pelayanan Kedokteran Tata Laksana Kanker Kolorektal: Kepmenkes RI Nomor HK.01.07/MENKES/406/2018. Kemenkes RI.
24 Jonasch E. 2001. Interferon in oncological practice: review of interferon biology, clinical applications, and toxicities. Oncologist 6: 34-55.   DOI
25 Abbas HT, Kyla-Nikkila K, Ra R, Saris PEJ. 2006. Nisin induction without nisin secretion. Microbiology 152: 1489-1496.   DOI
26 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.   DOI
27 Zhuang Z, Wu Z, Chen M, Wang PG. 2008. Secretion of human interferon-β 1b by recombinant Lactococcus lactis. Biotechnol. Lett. 30: 1819-1823.   DOI
28 Bermudez-Humaran 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.   DOI
29 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).
30 Wang W, Roberts CJ. 201. Protein aggregation-Mechanisms, detection, and control. Int. J. Pharm. 550: 251-268.   DOI
31 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.   DOI
32 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.   DOI
33 Kumaran J, Wei L, Kotra LP, Fish EN. 2007. A structural basis for interferon-α-receptor interactions. FASEB J. 21: 3288-32967.   DOI
34 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.
35 Bazhanova ED. 2005. Participation of interferon-alpha in regulation of apoptosis. J. Evol. Biochem. Physiol. 41: 127-133.   DOI
36 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.   DOI
37 Bayat O, Baradaran A, Ariff A, Mohamad R, Rahim RA. 2014. Intra-cellular production of IFN-alpha 2b in Lactococcus lactis. Biotechnol. Lett. 36: 581-585.   DOI
38 Borrero J, Jimenez JJ, Gutiez L, Herranz C, Cintas LM, Hernandez 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.   DOI
39 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.   DOI
40 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.   DOI
41 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.   DOI
42 Kementerian Kesehatan Republik Indonesia. 2015. Rencana Strategis Kementerian Kesehatan Tahun 2015-2019, Kepmenkes RI Nomor HK.02.02/Menkes/52/2015. Kemenkes RI. 248.
43 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: 2881-2890.
44 Badan Pengawasan Obat dan Makanan Republik Indonesia. 2005. Peraturan Badan pengawas Obat dan Makanan Republik Indonesia. Badan Pengawas Obat. dan. Makanan. 53: 1689-1699.
45 Ningrum RA. 2017. Human interferon Alpha2a as anti Hepatitis B and C. Indones. J. Clin. Pharm. 6: 298-310.   DOI
46 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.   DOI
47 Gull I, Samra ZQ, Aslam MS, Athar MA. 2013. Heterologous expression, immunochemical and computational analysis of recombinant human interferon alpha 2b. Springerplus 2: 264.   DOI
48 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.   DOI
49 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.   DOI
50 World Health Organization. 2019. Indonesia source GLOBOCAN 2018. Int. Agency Res. Cancer. 256: 1-2.