Login Register Cart 0
Generic placeholder image

Current Pharmaceutical Biotechnology

Editor-in-Chief

ISSN (Print): 1389-2010
ISSN (Online): 1873-4316

Back
Journal Home About Journal Editorial Board Journal Insight Current Issue Volumes/Issues
Subscribe
Research Article

Recombinant Expression of Cec-B Peptide in Escherichia coli with a Significant Anticancer Effect on Hepatocellular Carcinoma

Author(s): Hend Okasha, Sami Mohamed Nasr and Safia Samir*

Volume 22, Issue 9, 2021

Published on: 04 January, 2021

Page: [1235 - 1245] Pages: 11

DOI: 10.2174/1389201022666210104121709

Price: $65

Abstract

Background: Cecropin-B (Cec-B) is an Antimicrobial Peptide (AMP) found in insects.

Objectives: Recombinant production of Cec-B peptide in Escherichia coli (Rosetta™ DE3), and studying its anticancer effect on Hepatocellular Carcinoma Cell line (HCC).

Methods: The Cec-B gene of Drosophila melanogaster was synthesized by PCR assembly using the Simplified Gene Synthesis (SGS) method. To express the recombinant peptide in E. coli (Rosetta™ DE3); the synthesized gene was cloned into pET-15b expression vector. The recombinant peptide was expressed as insoluble aggregates called Inclusion Bodies (IBs) using 2mM lactose inducer. IBs were solubilized in a denatured form using 8 M urea followed by in-vitro protein refolding using rapid dilution method. The refolded Cec-B was purified using cation-exchange SP-FF column. Cytotoxicity of recombinant Cec-B (rCec-B) was reported on normal human lung cell line (WI-38), and Hepatocellular carcinoma cell line (HepG2).

Results: The Cec-B gene was expressed and purified at concentration 1.212±0.1 mg/ml which represents 48.49±4% of the total proteins injected to the column (2.5±0.2 mg/ml). The safe dose of purified rCec-B on normal WI-38 cells was calculated to be 1.57 mg/ml. The half-maximal Inhibitory Concentration (IC50) of rCec-B on HepG2 cell line was calculated to be 25 μg/ml. Scanning Electron Microscope (SEM) showed that untreated and treated HepG2 cells had cell diameters from 11-12.92 μm and 14.18-21.58 μm, respectively.

Conclusion: The results of this study revealed a successful expression of the rCec-B peptide using a pET-based expression system with a simple purification step. The purified peptide could be considered as a hopeful anticancer drug against HCC.

Keywords: Cecropin-B, recombinant expression, peptide, Escherichia coli, hepatocellular carcinoma, anticancer peptide.

« Previous Next »
Graphical Abstract

[1]
Morsi, E.A.; Ahmed, H.O.; Abdel-Hady, H.; El-Sayed, M.; Shemis, M.A. GC-analysis, Antioxidant, anti-inflammatory, and anticancer activities of some extracts and fractions of Linum usitatissimum. Curr. Bioact. Compd., 2020, 16(1), 1306-1318.http://www.eurekaselect.com/179040/article
[2]
Holohan, C.; Van Schaeybroeck, S.; Longley, D.B.; Johnston, P.G. Cancer drug resistance: An evolving paradigm. Nat. Rev. Cancer, 2013, 13(10), 714-726.http://www.nature.com/articles/nrc3599
[http://dx.doi.org/10.1038/nrc3599] [PMID: 24060863]
[3]
Chakraborty, S.; Rahman, T. The difficulties in cancer treatment. Ecancermedicalscience, 2012, 6, ed16.
[PMID: 24883085]
[4]
Falzone, L.; Salomone, S.; Libra, M. Evolution of cancer pharmacological treatments at the turn of the third millennium. Front. Pharmacol., 2018, 9, 1300.
[http://dx.doi.org/10.3389/fphar.2018.01300] [PMID: 30483135]
[5]
Costa, J.; Silva, N.; Sarmento, B.; Pintado, M. Delivery systems for antimicrobial peptides and proteins: Towards optimization of bioavailability and targeting. Curr. Pharm. Biotechnol., 2017, 18(2), 108-120.www.eurekaselect.com/openurl/content.php?genre=article&issn=1389-2010&volume=18&issue=2&spage=108
[6]
Bhopale, G.M. Antimicrobial peptides: A promising avenue for human healthcare. Curr. Pharm. Biotechnol., 2020, 21(2), 90-96.http://www.eurekaselect.com/175581/article
[http://dx.doi.org/10.2174/1389201020666191011121722]
[7]
Hancock, R.E.W.; Haney, E.F.; Gill, E.E. The immunology of host defence peptides: Beyond antimicrobial activity. Nat. Rev. Immunol., 2016, 16(5), 321-334.
[http://dx.doi.org/10.1038/nri.2016.29] [PMID: 27087664]
[8]
Shoombuatong, W.; Schaduangrat, N.; Nantasenamat, C. Unraveling the bioactivity of anticancer peptides as deduced from machine learning. EXCLI J., 2018, 17, 734-752.
[PMID: 30190664]
[9]
Sadatmousavi, P.; Soltani, M.; Nazarian, R.; Jafari, M.; Chen, P. Self-assembling peptides: Potential role in tumor targeting. Curr. Pharm. Biotechnol., 2011, 12(8), 1089-1100.www.eurekaselect.com/openurl/content.php?genre=article&issn=1389-2010&volume=12&issue=8&spage=1089
[10]
Boohaker, R.J.; Lee, M.W.; Vishnubhotla, P.; Perez, J.M.; Khaled, A.R. The use of therapeutic peptides to target and to kill cancer cells. Curr. Med. Chem., 2012, 19(22), 3794-3804.
[http://dx.doi.org/10.2174/092986712801661004] [PMID: 22725698]
[11]
Ekengren, S.; Hultmark, D. Drosophila cecropin as an antifungal agent. Insect Biochem. Mol. Biol., 1999, 29(11), 965-972.
[http://dx.doi.org/10.1016/S0965-1748(99)00071-5] [PMID: 10560137]
[12]
Suttmann, H.; Retz, M.; Paulsen, F.; Harder, J.; Zwergel, U.; Kamradt, J. Antimicrobial peptides of the Cecropin-family show potent antitumor activity against bladder cancer cells. BMC Urol., 2008, 8(1), 5.https://bmcurol.biomedcentral.com/articles/10.1186/1471-2490-8-5
[http://dx.doi.org/10.1186/1471-2490-8-5]
[13]
Andrä, J.; Berninghausen, O.; Leippe, M. Cecropins, antibacterial peptides from insects and mammals, are potently fungicidal against Candida albicans. Med. Microbiol. Immunol. (Berl.), 2001, 189(3), 169-173.http://link.springer.com/10.1007/s430-001-8025-x
[http://dx.doi.org/10.1007/s430-001-8025-x] [PMID: 11388616]
[14]
Hoskin, D.W.; Ramamoorthy, A. Studies on anticancer activities of antimicrobial peptides; Biochimica et Biophysica Acta - Biomembranes. NIH Public Access, 2008, Vol. 1778, 357-375.
[15]
Moore, A.J.; Devine, D.A.; Bibby, M.C. Preliminary experimental anticancer activity of cecropins. Pept. Res., 1994, 7(5), 265-269.
[PMID: 7849420]
[16]
Winder, D.; Günzburg, W.H.; Erfle, V.; Salmons, B. Expression of antimicrobial peptides has an antitumour effect in human cells. Biochem. Biophys. Res. Commun., 1998, 242(3), 608-612.
[http://dx.doi.org/10.1006/bbrc.1997.8014] [PMID: 9464264]
[17]
Isidro-Llobet, A.; Kenworthy, M.N.; Mukherjee, S.; Kopach, M.E.; Wegner, K.; Gallou, F. Sustainability challenges in peptide synthesis and purification: From R D to production. J. Org. Chem., 2019, 84(8), 4615-4628.https://pubs.acs.org/doi/10.1021/acs.joc.8b03001
[18]
Hou, W.; Zhang, X.; Liu, C.F. Progress in chemical synthesis of peptides and proteins. Transactions of Tianjin University, 2017, 23, 401-419.
[http://dx.doi.org/10.1007/s12209-017-0068-8]
[19]
Mueller, L.K.; Baumruck, A.C.; Zhdanova, H.; Tietze, A.A. Challenges and perspectives in chemical synthesis of highly hydrophobic peptides. Front. Bioeng. Biotechnol., 2020, 8, 162.https://www.frontiersin.org/article/10.3389/fbioe.2020.00162/full
[http://dx.doi.org/10.3389/fbioe.2020.00162] [PMID: 32195241]
[20]
Rodríguez, V.; Asenjo, J.A.; Andrews, B.A. Design and implementation of a high yield production system for recombinant expression of peptides. Microb. Cell Fact., 2014, 13(1), 65.
[http://dx.doi.org/10.1186/1475-2859-13-65] [PMID: 24885242]
[21]
Tutar, L.; Tutar, Y. Heat shock proteins; An overview. Curr. Pharm. Biotechnol., 2010, 11(2), 216-222.www.eurekaselect.com/openurl/content.php?genre=article&issn=1389-2010&volume=11&issue=2&spage=216
[22]
Tu, P.; Ma, Z.; Wang, H.; Ma, B.; Li, X.; Duan, H. Expression of CTB-10×rolGLP-1 in E. coli and its therapeutic effect on type 2 diabetes. Curr. Pharm. Biotechnol., 2015, 16(6), 564-572.www.eurekaselect.com/openurl/content.php?genre=article&issn=1389-2010&volume=16&issue=6&spage=564
[23]
Wei, Q.; Kim, Y.S.; Seo, J.H.; Jang, W.S.; Lee, I.H.; Cha, H.J. Facilitation of expression and purification of an antimicrobial peptide by fusion with baculoviral polyhedrin in Escherichia coli. Appl. Environ. Microbiol., 2005, 71(9), 5038-5043.
[http://dx.doi.org/10.1128/AEM.71.9.5038-5043.2005] [PMID: 16151084]
[24]
Kamionka, M. Engineering of therapeutic proteins production in Escherichia coli. Curr. Pharm. Biotechnol., 2011, 12(2), 268-274.
[http://dx.doi.org/10.2174/138920111794295693] [PMID: 21050165]
[25]
Okasha, H.; Samir, S. Synthesis and molecular cloning of antimicrobial peptide chromogranin A N-46 gene using conventional PCR. Gene Rep., 2020, 18, 100571.https://linkinghub.elsevier.com/retrieve/pii/S2452014419302134
[http://dx.doi.org/10.1016/j.genrep.2019.100571]
[26]
Lodish, H; Berk, A; Zipursky, SL; Matsudaira, P; Baltimore, D; Darnell, J DNA Cloning with Plasmid Vectors, 2000.
[27]
Froger, A.; Hall, J.E. Transformation of plasmid DNA into E. coli using the heat shock method. J. Vis. Exp., 2007, 1(6)www.jove.com/index/Details.stp?ID=253
[28]
Liao, M. cDNA cloning of growth hormone from giant panda (Ailuropoda melanoleuca) and its expression in Escherichia coli. Comp. Biochem. Physiol. Part B., 2003, 135(1), 109-116.https://linkinghub.elsevier.com/retrieve/pii/S1096495903000563
[29]
Das, A.; Verma, A.; Mukherjee, K.J. Synthesis of dopamine in E. coli using plasmid-based expression system and its marked effect on host growth profiles. Prep. Biochem. Biotechnol., 2017, 47(8), 754-760.https://www.tandfonline.com/doi/full/10.1080/10826068.2017.1320291
[http://dx.doi.org/10.1080/10826068.2017.1320291] [PMID: 28430025]
[30]
Palmer, I.; Wingfield, P.T. Preparation and extraction of insoluble (Inclusion-body) proteins from Escherichia coli. Curr. Protoc. Protein Sci., 2012, 1 (SUPPL.70).
[31]
Li, R.; Zhu, L-N.; Ren, L-Q.; Weng, J-Y.; Sun, J-S. Molecular cloning and characterization of glycogen synthase in Eriocheir sinensis. Comp. Biochem. Physiol. Part B Biochem. Mol. Biol., 2017, 47-56.https://linkinghub.elsevier.com/retrieve/pii/S1096495917301422
[http://dx.doi.org/10.1016/j.cbpb.2017.09.004]
[32]
Singh, A.; Upadhyay, V.; Panda, A.K. Solubilization and refolding of inclusion body proteins. Methods Mol. Biol., 2015, 1258, 283-291.
[http://dx.doi.org/10.1007/978-1-4939-2205-5_15] [PMID: 25447870]
[33]
Tischer, A.; Lilie, H.; Rudolph, R.; Lange, C. L-arginine hydrochloride increases the solubility of folded and unfolded recombinant plasminogen activator rPA. Protein Sci., 2010, 19(9), 1783-1795.
[http://dx.doi.org/10.1002/pro.465] [PMID: 20665695]
[34]
Tsumoto, K.; Arakawa, T.; Chen, L. Step-wise refolding of recombinant proteins. Curr. Pharm. Biotechnol., 2010, 11(3), 285-288.http://www.eurekaselect.com/openurl/content.php?genre=article&issn=1389-2010&volume=11&issue=3&spage=285
[http://dx.doi.org/10.2174/138920110791111979]
[35]
Duong-Ly, K.C.; Gabelli, S.B. Using ion exchange chromatography to purify a recombinantly expressed protein. Methods Enzymol., 2014, 541, 95-103.
[http://dx.doi.org/10.1016/B978-0-12-420119-4.00008-2]
[36]
Zhang, X.; Yoon, H.J.; Kang, M.G.; Kim, G.J.; Shin, S.Y.; Baek, S.H.; Lee, J.G.; Bai, J.; Lee, S.Y.; Choi, M.J.; Hong, K.; Bae, H. Identification and evaluation of cytotoxicity of peptide liposome incorporated citron extracts in an in vitro system. Int. J. Mol. Sci., 2018, 19(2), 626.
[http://dx.doi.org/10.3390/ijms19020626] [PMID: 29470444]
[37]
Vijayarathna, S.; Sasidharan, S. Cytotoxicity of methanol extracts of Elaeis guineensis on MCF-7 and Vero cell lines. Asian Pac. J. Trop. Biomed., 2012, 2(10), 826-829.
[http://dx.doi.org/10.1016/S2221-1691(12)60237-8] [PMID: 23569855]
[38]
Nan, K-J.; Cui, J.; Guo, Y-H.; Zhang, H-Y.; Jiang, L-L.; Ma, J-Q. Cyclooxygenase-2 inhibitor is a robust enhancer of anticancer agents against hepatocellular carcinoma multicellular spheroids. Onco Targets Ther, 2014, 7, 353.http://www.dovepress.com/cyclooxygenase-2-inhibitor-is-a-robust-enhancer-of-anticancer-agents-a-peer-reviewed-article-OTT
[http://dx.doi.org/10.2147/OTT.S56115]
[39]
Yue, Y.; Yang, Y.; Shi, L.; Wang, Z. Suppression of human hepatocellular cancer cell proliferation by Brucea javanica oil-loaded liposomes via induction of apoptosis. Arch. Med. Sci., 2015, 11(4), 856-862.
[http://dx.doi.org/10.5114/aoms.2015.53306] [PMID: 26322098]
[40]
Thundimadathil, J. Cancer treatment using peptides: Current therapies and future prospects. J. Amino Acids, 2012, 2012, 967347.
[http://dx.doi.org/10.1155/2012/967347] [PMID: 23316341]
[41]
Jin, X.; Mei, H.; Li, X.; Ma, Y.; Zeng, A.H.; Wang, Y.; Lu, X.; Chu, F.; Wu, Q.; Zhu, J. Apoptosis-inducing activity of the antimicrobial peptide cecropin of Musca domestica in human hepatocellular carcinoma cell line BEL-7402 and the possible mechanism. Acta Biochim. Biophys. Sin. (Shanghai), 2010, 42(4), 259-265.
[http://dx.doi.org/10.1093/abbs/gmq021] [PMID: 20383464]
[42]
Dubey, S.; Mody, N.; Sharma, R.; Agrawal, U.; Vyas, S.P. Nanobiomaterials: Novel nanoplatforms for protein and peptide delivery. In:Nanobiomaterials in Drug Delivery; William Andrew Publishing, 2016, pp. 111-146.https://linkinghub.elsevier.com/retrieve/pii/B9780323428668000046
[http://dx.doi.org/10.1016/B978-0-323-42866-8.00004-6]
[43]
Sinha, R.; Shukla, P. Antimicrobial peptides: Recent insights on biotechnological interventions and future perspectives. Protein Pept. Lett., 2019, 26(2), 79-87.http://www.eurekaselect.com/166684/article
[http://dx.doi.org/10.2174/0929866525666181026160852] [PMID: 30370841]
[44]
Yan, J.; Zhao, S.F.; Mao, Y.F.; Luo, Y.H. Effects of lactose as an inducer on expression of Helicobacter pylori rUreB and rHpaA, and Escherichia coli rLTKA63 and rLTB. World J. Gastroenterol, 2004, 10(12), 1755-1758.http://www.wjgnet.com/1007-9327/full/v10/i12/1755.htm
[http://dx.doi.org/10.3748/wjg.v10.i12.1755] [PMID: 15188500]
[45]
Sambrook, J.; Russell, D.W. Molecular cloning: A laboratory manual, 3rd ed; Zool. Res, 2001.
[46]
Grönberg, A. Ion Exchange Chromatography. Biopharmaceutical Processing; Elsevier, 2018, pp. 379-399.https://linkinghub.elsevier.com/retrieve/pii/B9780081006238000189
[http://dx.doi.org/10.1016/B978-0-08-100623-8.00018-9]
[47]
Pergande, M.R.; Cologna, S.M. Isoelectric point separations of peptides and proteins. Proteomes, 2017, 5(1), 4.http://www.mdpi.com/2227-7382/5/1/4
[http://dx.doi.org/10.3390/proteomes5010004] [PMID: 28248255]
[48]
Eriksson, K.O. Reversed Phase Chromatography. In:Biopharmaceutical Processing; Elsevier Science B. V: Amsterdam, 2018, pp. 433-439.
[http://dx.doi.org/10.1016/B978-0-08-100623-8.00022-0]
[49]
Williams, A.; Frasca, V. Ion-exchange chromatography. Curr. Protoc. Protein Sci., 1999, 15(1)https://onlinelibrary.wiley.com/doi/abs/10.1002/0471140864.ps0802s15
[http://dx.doi.org/10.1002/0471140864.ps0802s15] [PMID: 18429204]
[50]
Stokes, D.J. Principles and practice of Variable Pressure/ Environmental Scanning Electron Microscopy (VP-ESEM). Principles and Practice of Variable Pressure/Environmental Scanning Electron Microscopy (VP-ESEM)., 2008.doi.wiley.com/10.1002/9780470758731

Rights & Permissions Print Cite
Article Metrics
29
1
© 2024 Bentham Science Publishers | Privacy Policy