Modification of recombinant human epidermal growth factor (rh-EGF) expression vector by site directed mutagenesis for therapeutic protein production

https://doi.org/10.22146/ijbiotech.41859

Achmad Rodiansyah(1*), Riyona Desvy Pratiwi(2), Sabighoh Zanjabila(3), Asrul M. Fuad(4)

(1) Department of Biology, State University of Malang, Semarang Street No. 5, Malang, 65145, Indonesia; Research Center for Biotechnology, Indonesian Institute of Sciences, Jalan Raya Bogor KM.46, Cibinong, 16911, Indonesia
(2) Research Center for Biotechnology, Indonesian Institute of Sciences, Jalan Raya Bogor KM.46, Cibinong, 16911, Indonesia
(3) Research Center for Biotechnology, Indonesian Institute of Sciences, Jalan Raya Bogor KM.46, Cibinong, 16911, Indonesia
(4) Research Center for Biotechnology, Indonesian Institute of Sciences, Jalan Raya Bogor KM.46, Cibinong, 16911, Indonesia
(*) Corresponding Author

Abstract


Recombinant human epidermal growth factor (rh-EGF) has high value in therapies for h-EGF deficiency related 10 diseases. The recombinant protein was designed to be expressed in pET21b(+) vector using Escherichia coli 11 BL21(DE3) expression host. However, in our previous study, the gene of rh-EGF was constructed aside gene of 12 6xHisTag without any restriction sites, so it is not possible to obtained a purified and single rh-EGF. In this study, 13 we modified the rh-EGF expression vector with a common method, namely site-directed mutagenesis (SDM) to 14 remove the gene of 6xHisTag. The vector modification was carried out by inserting stop codons and EcoR1 15 restriction site, also deleting 6xHisTag sequence with PCR-based SDM. The results of PCR showed non-specific 16 bands, PCR 2-step cycles produced one non-specific band and PCR 3-step cycles produced two non-specific 17 bands. All of the PCR products were purified by gene isolation. The SDM-recombinant plasmids which were 18 treated for template plasmid-free product were transformed to E.coli DH5α. The result of transformation had low 19 transformant efficiency score. However, gene mutation with deletion of 6xHisTag and insertion of stop codons 20 and EcoRI restriction site in plasmid pET21b(+) had been successfully done. In addition, in the expression level, 21 it is proven that the modified vector results rh-EGF which has similar size with rh-EGF standard and 22 approximately 1 kDa smaller than that of our previous rh-EGF-6xHisTag. 23


Keywords


recombinant human epidermal growth factor (rh-EGF); site-directed mutagenesis (SDM); pET21b(+); Escherichia coli BL21(DE3)

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References

Antikainen NM, Martin SF. 2005. Altering protein specificity: techniques and applications. Bioorg Med Chem. 13(8):2701–2716. doi:10.1016/j.bmc.2005.01.059.

Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K. 2002. Short protocols in molecular biology. 5th ed. New York: John Wiley & Sons.

Balagurumoorthy P, Adelstein SJ, Kassis AI. 2008. Method to eliminate linear DNA from mixture containing nicked-circular, supercoiled, and linear plasmid DNA. Anal Biochem. 381(1):172–174. doi:10.1016/j.ab.2008.06.037.

Bell GI, Fong NM, Stempien MM, Wormsted MA, Caput D, Ku LL, Urdea MS, Rall LB, Sanchez-Pescador R. 1986. Human epidermal growth factor precursor: cDNA sequence, expression in vitro and gene organization. Nucleic Acids Res. 14(21):8427–8446.

Bhatia S. and Dahiya R. 2015. Concepts and techniques of plant tissue culture science. In: Modern Applications of Plant Biotechnology in Pharmaceutical Sciences. Elsevier. p. 121–156.

Biolabs. 2019. Q5®  Site-directed mutagenesis kit (without competent cells). New England Biolabs, Inc.

Carbone A, Fioretti FM, Fucci L, Ausió J, Piscopo M. 2012. High efficiency method to obtain supercoiled DNA with a commercial plasmid purification kit. Acta Biochim Pol. 59(2):275–278.

Carson S, Miller H, Witherow DS. 2012. Replica plating. In: Molecular Biology Techniques A Classroom Laboratory Manual (Third Edition). Academic Press.

Cebrián J, Kadomatsu-Hermosa MJ, Castán A, Martínez V, Parra C, Fernández-Nestosa MJ, Schaerer C, Martínez-Robles M-L, Hernández P, Krimer DB, et al. 2015. Electrophoretic mobility of supercoiled, catenated and knotted DNA molecules. Nucleic Acids Res. 43(4):e24. doi:10.1093/nar/gku1255.

Chang AY, Chau VW, Landas JA, Pang Y. 2017. Preparation of calcium competent Escherichia coli and heat-shock transformation. JEMI Methods. 1:22–25.

Citri A. and Yarden Y. 2006. EGF–ERBB signalling: towards the systems level. Nature Reviews Molecular Cell Biology. 7(7):505–516. doi:10.1038/nrm1962.

Das S. and Dash HR. 2015. Cloning and transformation. In: Das S, Dash HR, editors. Microbial biotechnology- a laboratory manual for bacterial systems. New Delhi: Springer India. p. 35–72.

Dvorak B. 2010. Milk epidermal growth factor and gut protection. J Pediatr. 156(2 Suppl):S31-35. doi:10.1016/j.jpeds.2009.11.018.

Edelheit O, Hanukoglu A, Hanukoglu I. 2009. Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies. BMC Biotechnol. 9:61. doi:10.1186/1472-6750-9-61.

Fisher DA. and Lakshmanan J. 1990. Metabolism and effects of epidermal growth factor and related growth factors in mammals. Endocr Rev. 11(3):418–442. doi:10.1210/edrv-11-3-418.

Flamant M, Bollée G, Hénique C, Tharaux P-L. 2012. Epidermal growth factor: a new therapeutic target in glomerular disease. Nephrol Dial Transplant. 27(4):1297–1304. doi:10.1093/ndt/gfs030.

Francis MS, Amer AAA, Milton DL, Costa TRD. 2017. Site-directed mutagenesis and its application in studying the interactions of T3S components. Methods Mol Biol. 1531:11–31. doi:10.1007/978-1-4939-6649-3_2.

Fu X, Sun X, Sun T, Dong Y, Gu X, Chen W, Sheng Z-Y. 2002. Epidermal growth factor stimulates tissue repair in skin through skin stem cell activation. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 16(1):31–35.

Geneaid. 2017. Gel/PCR DNA fragments kit (DF100, DF300) | Geneaid.

Higashiyama S, Iwabuki H, Morimoto C, Hieda M, Inoue H, Matsushita N. 2008. Membrane-anchored growth factors, the epidermal growth factor family: beyond receptor ligands. Cancer Sci. 99(2):214–220. doi:10.1111/j.1349-7006.2007.00676.x.

Hochuli A, Bannwarth W, Dobeli H, Stuber D. 1988. Genetic approach to facilitate purification  of recombinant proteins with a novel metal chelate adsorbent. Nature Publishing Group.(350):1321–1325.

Huang G, Besner GE, Brigstock DR. 2012. Heparin-binding epidermal growth factor-like growth factor suppresses experimental liver fibrosis in mice. Lab Invest. 92(5):703–712. doi:10.1038/labinvest.2012.3.

Johnston C, Polard P, Claverys J-P. 2013. The DpnI/DpnII pneumococcal system, defense against foreign attack without compromising genetic exchange. Mob Genet Elements. 3(4). doi:10.4161/mge.25582.

Joko T, Kusumandari N, Hartono S. 2011. [Optimasi metode PCR untuk deteksi                       Pectobacterium carotovorum, penyebab penyakit busuk lunak Anggrek]. Jurnal Perlindungan Tanaman Indonesia. 17(2):54–59. doi:10.22146/jpti.9813.

Jones PA. 2012. Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat Rev Genet. 13(7):484–492. doi:10.1038/nrg3230.

Kalendar R, Tselykh TV, Khassenov B, Ramanculov EM. 2017. Introduction on Using the FastPCR Software and the Related Java Web Tools for PCR and Oligonucleotide Assembly and Analysis. Methods Mol Biol. 1620:33–64. doi:10.1007/978-1-4939-7060-5_2.

Kharchenko MV, Aksyonov AA, Melikova MM, Kornilova ES. 2007. Epidermal growth factor (EGF) receptor endocytosis is accompanied by reorganization of microtubule system in HeLa cells. Cell Biol Int. 31(4):349–359. doi:10.1016/j.cellbi.2007.01.020.

Klein J, Bascands J-L, Buffin-Meyer B, Schanstra JP. 2016. Epidermal growth factor and kidney disease: a long-lasting story. Kidney Int. 89(5):985–987. doi:10.1016/j.kint.2016.02.020.

Lohman GJS, Zhang Y, Zhelkovsky AM, Cantor EJ, Evans TC. 2014. Efficient DNA ligation in DNA-RNA hybrid helices by Chlorella virus DNA ligase. Nucleic Acids Res. 42(3):1831–1844. doi:10.1093/nar/gkt1032.

Lorenz TC. 2012. Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies. J Vis Exp.(63):e3998. doi:10.3791/3998.

Mattos JCPD, Dantas FJS, Caldeira‐de‐Araújo A, Moraes MO. 2004. Agarose gel electrophoresis system in the classroom: Detection of DNA strand breaks through the alteration of plasmid topology. Biochemistry and Molecular Biology Education. 32(4):254–257. doi:10.1002/bmb.2004.494032040382.

Moore LD, Le T, Fan G. 2013. DNA methylation and its basic function. Neuropsychopharmacology. 38(1):23–38. doi:10.1038/npp.2012.112.

Nair RR, Warner BB, Warner BW. 2008. Role of epidermal growth factor and other growth factors in the prevention of necrotizing enterocolitis. Semin Perinatol. 32(2):107–113. doi:10.1053/j.semperi.2008.01.007.

Nurmalasari. 2010. [Konstruksi gen sintetik EGFsyn pengkode human epidermal growth factor (hEGF) menggunakan metode thermodinamically balanced insite-out (TBIO)]. [Depok]: Universitas Indonesia.

Osakabe K, Osakabe Y, Toki S. 2010. Site-directed mutagenesis in Arabidopsis using custom-designed zinc finger nucleases. Proc Natl Acad Sci USA. 107(26):12034–12039. doi:10.1073/pnas.1000234107.

Pikuła M, Langa P, Kosikowska P, Trzonkowski P. 2015. Stem cells and growth factors in wound healing. Postepy Hig Med Dosw. 69:874–885.

Putri DED. & Sriwidodo. 2016. [Peranan epidermal growth factor pada penyembuhan luka pasien ulkus diabetes]. Farmaka. 14(4):61–69. doi:10.24198/jf.v14i4.9531.

Rabhi I, Guedel N, Chouk I, Zerria K, Barbouche MR, Dellagi K, Fathallah DM. 2004. A novel simple and rapid PCR-based site-directed mutagenesis method. Mol Biotechnol. 26(1):27–34. doi:10.1385/MB:26:1:27.

Rahimzadeh M, Sadeghizadeh M, Najafi F, Arab S, Mobasheri H. 2016. Impact of heat shock step on bacterial transformation efficiency. Mol Biol Res Commun. 5(4):257–261.

Roche. 2012. Restriction endonuclease EcoRI.

Roux KH. 1995. Optimization and troubleshooting in PCR. PCR methods and applications. 4(5):NaN-NaN. doi:10.1101/gr.4.5.S185.

Sánchez-Romero MA, Cota I, Casadesús J. 2015. DNA methylation in bacteria: from the methyl group to the methylome. Curr Opin Microbiol. 25:9–16. doi:10.1016/j.mib.2015.03.004.

Savage CR, Hash JH, Cohen S. 1973. Epidermal growth factor. Location of disulfide bonds. J Biol Chem. 248(22):7669–7672.

Savage CR, Inagami T, Cohen S. 1972. The Primary Structure of Epidermal Growth Factor. J Biol Chem. 247(23):7612–7621. [accessed 2019 Apr 20]. http://www.jbc.org/content/247/23/7612.

She W, Ni J, Shui K, Wang F, He R, Xue J, Reetz MT, Li A, Ma L. 2018. Rapid and error-free site-directed mutagenesis by a PCR-free in vitro CRISPR/Cas9-mediated mutagenic system. ACS Synth Biol. 7(9):2236–2244. doi:10.1021/acssynbio.8b00245.

Shivanand P. & Noopur S. 2010. Recombinant DNA technology and genetic engineering: A safe and effective meaning for production valuable biologicals. International Journal of Pharmaceutical Sciences Review and Research. 1(1):14–20.

Sighn M, Yadav A, Ma X, Amoah E. 2010. Plasmid DNA transformation in Escherichia Coli : Effect of heat shock temperature, duration, and cold incubation of CaCl2 treated cells. International Journal of Biotechnology and Biochemistry. 6(4):561–568.

Su Z, Huang Y, Zhou Q, Wu Z, Wu X, Zheng Q, Ding C, Li X. 2006. High-level expression and purification of human epidermal growth factor with SUMO fusion in Escherichia coli. Protein Pept Lett. 13(8):785–792.

Suortti T. 1997. Coupled size-exclusion chromatography-anion-exchange chromatography in the analysis of poly- and oligosaccharides. Journal of Chromatography A. 763(1–2):331–335. doi:10.1016/S0021-9673(96)00996-X.

Tegel H, Ottosson J, Hober S. 2011. Enhancing the protein production levels in Escherichia coli with a strong promoter: Protein production levels in E. coli. FEBS Journal. 278(5):729–739. doi:10.1111/j.1742-4658.2010.07991.x.

Toyobo. KOD -Plus- Mutagenesis kit.

Vestheim H. and Jarman SN. 2008. Blocking primers to enhance PCR amplification of rare sequences in mixed samples – a case study on prey DNA in Antarctic krill stomachs. Frontiers in Zoology. 5(1):12. doi:10.1186/1742-9994-5-12.

Walquist MJ. and El‐Gewely MR. 2018. Mutagenesis: Site-Directed. In: eLS. American Cancer Society. p. 1–14.

Wan H, Li Y, Fan Y, Meng F, Chen C, Zhou Q. 2012. A site-directed mutagenesis method particularly useful for creating otherwise difficult-to-make mutants and alanine scanning. Anal Biochem. 420(2):163–170. doi:10.1016/j.ab.2011.09.019.

Wu HG, Song SY, Kim YS, Oh YT, Lee CG, Keum KC, Ahn YC, Lee S. 2009. Therapeutic effect of recombinant human epidermal growth factor (rhEGF) on mucositis in patients undergoing radiotherapy, with or without chemotherapy, for head and neck cancer. Cancer. 115(16):3699–3708. doi:10.1002/cncr.24414.

Xian CJ. 2007. Roles of epidermal growth factor family in the regulation of postnatal somatic growth. Endocr Rev. 28(3):284–296. doi:10.1210/er.2006-0049.

Xu Z, Colosimo A, Gruenert DC. 2003. Site-Directed Mutagenesis using the megaprimer method. In: Casali N, Preston A, editors. E. coli Plasmid Vectors: Methods and Applications. Totowa, NJ: Humana Press. (Methods in Molecular BiologyTM). p. 203–207.

Zeng F. and Harris RC. 2014. Epidermal growth factor, from gene organization to bedside. Semin Cell Dev Biol. 28:2–11. doi:10.1016/j.semcdb.2014.01.011.

Zhang B, Zhang X, An X, Ran D, Zhou Y, Lu J, Tong Y. 2009. An easy-to-use site-directed mutagenesis method with a designed restriction site for convenient and reliable mutant screening. J Zhejiang Univ Sci B. 10(6):479–482. doi:10.1631/jzus.B0820367.

Zhu H. and Qian J. 2012. Applications of functional protein microarrays in basic and clinical research. In: Advances in Genetics. Vol. 79. Elsevier. p. 123–155.



DOI: https://doi.org/10.22146/ijbiotech.41859

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