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

Meilina, Lita; Budiarti, Sri; Mustopa, Apon Zaenal; Darusman, Huda Shalahudin; Triratna, Lita; Nugraha, Muhammad Ajietuta; Bilhaq, Muhammad Sabiq; Ningrum, Ratih Asmana

doi:10.48022/mbl.2009.09008

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

Microbiology and biotechnology letters = 한국미생물·생명공학회지, v.49 no.1, 2021년, pp.75 - 87

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))

Abstract ▼ AI-Helper

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.

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표/그림 (10)

표 Table 1. Bacterial strains and plasmids used in this study.
그림 Fig. 1. Representation of the pNZ8148-SP_Usp45-IFNα-2b construct map. The recombinant plasmid containing insert gene IFNα- 2b and SP_Usp45 was concatenated and ligated into pNZ8148 expression vector.
그림 Fig. 2. Sequence alignment of codon-optimized and native IFNα-2b used Geneious Prime^® software. Line graph represented base contain, GC graph was pointed by blue line, and AT graph in green line. Gaps were found on different places (shown by black arrows). Geneious Prime^® software allowed to quantified GC contents percentage of codon optimized sequence (shown by green circle), which showed the amount of A, C, G, T, U, S, W nucleotides in the sequence, foremost G, C or S.
그림 Fig. 3. Predicted 3D structure of codon-optimized recombinant IFNα-2b using Phyre2 Server. The helices are starting from N-terminal, which are containing five alpha helices (A-E). Two disulfide bonds were pointed in yellow bonds for Cys 1-Cys 98 and Cys 29-Cys 138. Free cysteine residues were in single yellow atom color.
그림 Fig. 4. Validation of DNA plasmid construct in L. lactis recombinant IFNα-2b. (A) DNA plasmid PCR product electrophoregram used F/R PnisA and TpNZ8148 primers; (B) electrophoregram of isolated recombinant plasmid from transformant colonies 1-5. M: DNA marker of 100 bp (Vivantis).
그림 Fig. 5. BLAST tool confirmation of codon-optimized IFNα-2b amino acid sequence was 100% identitical to human IFNα-2b.
$Fig. 6. Chromatogram profile of Size Exclusion Chromatograph (SEC) IFNα-2b. (A) Absorbance of IFNα-2b measured at 280 nm; (B) Tricine SDS-PAGE of partial fraction 4-9; (C) Dot-blot hybridization of confirmed SEC fraction; (D) Western blot analysis of partial fraction 4-6 of recombinant L. lactis pNZ8148-SP<sub>Usp45</sub>-IFNα-2b; (+) control of rhIFNα-2 (Biovision # P01563).$ 그림 Fig. 6. Chromatogram profile of Size Exclusion Chromatograph (SEC) IFNα-2b. (A) Absorbance of IFNα-2b measured at 280 nm; (B) Tricine SDS-PAGE of partial fraction 4-9; (C) Dot-blot hybridization of confirmed SEC fraction; (D) Western blot analysis of partial fraction 4-6 of recombinant L. lactis pNZ8148-SP_Usp45-IFNα-2b; (+) control of rhIFNα-2 (Biovision # P01563).
$Fig. 7. Total protein quantification of IFNα-2b in every step of purification using BCA Kit Assay. Culture Supernatant (CS), Host (L. lactis NZ3900) Supernatant (HS), partial purified fraction 5 and 6 (F5-F6).$ 그림 Fig. 7. Total protein quantification of IFNα-2b in every step of purification using BCA Kit Assay. Culture Supernatant (CS), Host (L. lactis NZ3900) Supernatant (HS), partial purified fraction 5 and 6 (F5-F6).
표 Table 2. The cytotoxic effect in WiDr cell viability and IC₅₀ determination.
그림 Fig. 8. WiDr cells morphology after samples treatment at 400 μg/ml. (A) Untreated cells; (B) treated with CS, (C) dialysis; (D) F5; (E) F6; (F) cells treated with HS; (G) positive control of treated cells with tamoxifen.

참고문헌 (50)

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.

상세보기
World Health Organization. 2019. Indonesia source GLOBOCAN 2018. Int. Agency Res. Cancer. 256: 1-2.
Elsaleh H, Grieu F, Joseph DJ, Iacopetta B. 2001. Molecular epidemiology of colorectal cancer. Int. J. Radiat. Oncol. 51: 260-261.
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.

상세보기
Wells JM, Mercenier A. 2008. Mucosal delivery of therapeutic and prophylactic molecules using lactic acid bacteria. Nature 6: 349-362.
Ningrum RA. 2014. Human Interferon Alpha-2b: A Therapeutic Protein for cancer treatment. Scientifica 2014: 1-9.

상세보기
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.

상세보기
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.
Badan Pengawasan Obat dan Makanan Republik Indonesia. 2005. Peraturan Badan pengawas Obat dan Makanan Republik Indonesia. Badan Pengawas Obat. dan. Makanan. 53: 1689-1699.
Kementerian Kesehatan Republik Indonesia. 2015. Rencana Strategis Kementerian Kesehatan Tahun 2015-2019, Kepmenkes RI Nomor HK.02.02/Menkes/52/2015. Kemenkes RI. 248.
Ningrum RA. 2017. Human interferon Alpha2a as anti Hepatitis B and C. Indones. J. Clin. Pharm. 6: 298-310.

상세보기
Gull I, Samra ZQ, Aslam MS, Athar MA. 2013. Heterologous expression, immunochemical and computational analysis of recombinant human interferon alpha 2b. Springerplus 2: 264.

상세보기
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.

상세보기
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.

상세보기
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.

상세보기
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.
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.

원문보기 상세보기
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.

상세보기
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.

상세보기
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.

상세보기
Sambrook J, Green MR. 2012. Molecular Cloning: A Laboratory Manual. pp. 1616. 4th Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
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.

상세보기
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.
Kumaran J, Wei L, Kotra LP, Fish EN. 2007. A structural basis for interferon-α-receptor interactions. FASEB J. 21: 3288-32967.

상세보기
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.
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.

상세보기
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.

상세보기
Aitken A, Learmonth MP. 2002. Protein determination by UV absorption, the protein protocols handbook. pp. 3-6. 2nd Ed. Totowa, New Jersey.
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.
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.
Winikoff SE, Zeh HJ, Demarco R, Lotze MT. 2006. Cytolytic Assays. pp. 827-849. Pittsburgh, USA.
Meerloo J van, Kaspers GJ, Cloos J. 2011. Cancer cell culture, MTT assay. Methods Mol. Biol. 731: 79-91.

상세보기
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.

상세보기
Ekowati H, Astuti I, Mustofa M. 2010. Anticancer activity of calanone on HeLa cell line. Indones J. Chem. 10: 240-244.

상세보기
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.

상세보기
Bazhanova ED. 2005. Participation of interferon-alpha in regulation of apoptosis. J. Evol. Biochem. Physiol. 41: 127-133.

상세보기
Kementerian Kesehatan Republik Indonesia. 2018. Pedoman Nasional Pelayanan Kedokteran Tata Laksana Kanker Kolorektal: Kepmenkes RI Nomor HK.01.07/MENKES/406/2018. Kemenkes RI.
Jonasch E. 2001. Interferon in oncological practice: review of interferon biology, clinical applications, and toxicities. Oncologist 6: 34-55.

상세보기
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.

상세보기
Abbas HT, Kyla-Nikkila K, Ra R, Saris PEJ. 2006. Nisin induction without nisin secretion. Microbiology 152: 1489-1496.

상세보기
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.

상세보기
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.

상세보기
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.

상세보기
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.

상세보기
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.

상세보기
Zhuang Z, Wu Z, Chen M, Wang PG. 2008. Secretion of human interferon-β 1b by recombinant Lactococcus lactis. Biotechnol. Lett. 30: 1819-1823.

상세보기
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.

상세보기
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.

상세보기
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).
Wang W, Roberts CJ. 201. Protein aggregation-Mechanisms, detection, and control. Int. J. Pharm. 550: 251-268.

상세보기

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