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http://dx.doi.org/10.5762/KAIS.2020.21.9.559

Expression of Recombinant Human Epidermal Growth Factor as a Active Form through Codon Optimization with E. coli and Co-expression of Chaperone  

Jang, Eun-Bin (Department of Biomedical Materials, Konyang University)
Kim, Jun Su (Department of Biomedical Materials, Konyang University)
Lee, Woo-Yiel (Department of Biomedical Materials, Konyang University)
Publication Information
Journal of the Korea Academia-Industrial cooperation Society / v.21, no.9, 2020 , pp. 559-568 More about this Journal
Abstract
Epidermal growth factor (EGF) is a hormone protein that affects cell growth and proliferation, and has various medical applications. In the present study, the gene of human EGF was codon-optimized with E. coli and the expression vector was constructed by cloning into pRSET. In order to obtain the recombinant human EGF in an active form rather than an inclusion body, chaperone co-expression was attempted along with codon optimization, for the first time. The expressed human EGF was isolated in the pure form by performing Ion Exchange Chromatography in two consecutive runs. ELISA analysis showed that the activity of purified EGF was greater than 99%, which is similar to commercially available EGF. Cell proliferation test confirmed that the recombinant human EGF has the ability to promote cell proliferation of human skin fibroblasts. The human EGF expression system of this study gives a significant amount of protein, and does not require the renaturation step and the additional chromatographic system to remove a fusion contaminant, thereby providing a very useful alternative to conventional expression systems for the preparation of recombinant human EGF.
Keywords
Epidermal Growth Factor; Recombination; Co-Expression; Codon Optimization; Chaperone;
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1 Su, Z., Huang, Y., Zhou, Q., Wu, Z., Wu, X., Zheng, Q., Ding, C., & Li, X. "High-Level Expression and Purification of Human Epidermal Growth Factor with SUMO Fusion in Escherichia coli", Protein & Peptide Letters, 13(8), 785-792. (2006). DOI: https://doi.org/10.2174/092986606777841280   DOI
2 Heejin Jeong, "Fusion protein synthesis technology development and research trend", BRIC View 2017-T38(2017). http://www.ibric.org/myboard/read.php?Board=report&id=2849 (Oct.26, 2017)
3 Yujin E. Kim, Mark S. Hipp, Andreas Bracher, Manajit Hayer-Hartl, and F. Ulrich Hartl "Molecular Chaperone Functions in Protein Folding and Proteostasis", This article's DOI: https://doi.org/10.1146/annurev-biochem-060208-09 2442 (2013)
4 Hartl, F. U. "Molecular chaperones in cellular protein folding", Nature, 381(6583), 571-580. (1996). DOI: https://doi.org/10.1038/381571a0   DOI
5 Bukau, B., & Horwich, A. L. "The Hsp70 and Hsp60 chaperone machines", Cell, 92(3), 351-366. (1998). DOI: https://doi.org/10.1016/S0092-8674(00)80928-9   DOI
6 Frydman, J. "Folding of Newly Translated Proteins In Vivo: The Role of Molecular Chaperones", Annual Review of Biochemistry, 70(1), 603-647. (2001). DOI: https://doi.org/10.1146/annurev.biochem.70.1.603   DOI
7 Hartl, F. U., & Hayer-Hartl, M. "Molecular chaperones in the cytosol: From nascent chain to folded protein", Science, 295(5561), 1852-1858. (2002). DOI: https://doi.org/10.1126/science.1068408   DOI
8 Kwon, M., Park, S., Kim, B., Kim, S., Nam, S., Society, K., Life, O., & Information, C. "Improvement of production of active cyclodextrin glucanotransferase by coexpression GroEL/ES chaperone in E. coli", Korean Society Of Life Science (2002).
9 Suk Young Kim, "The Role of Heat Shock Protein in Perinatal Fields", Korean J Perinatol Vol. 16, No. 1(2005).
10 Shuo-Shuo, C., Xue-Zheng, L., & Ji-Hong, S. "Effects of co-expression of molecular chaperones on heterologous soluble expression of the cold-active lipase", Lip-948. Protein Expression and Purification, 77(2), 166-172. (2011). DOI: https://doi.org/10.1016/j.pep.2011.01.009   DOI
11 Soung-Hun Roh, Corey F. Hryc, Hyun-Hwan Jeong, Xue Fei, Joanita Jakana, George H. Lorimer, and Wah Chiu, "Subunit conformational variation within individual GroEL oligomers resolved by Cryo-EM", Proc Natl Acad Sci U S A, 114(31): 8259-8264, 2017 Aug 1. DOI: https://doi.org/10.1073/pnas.1704725114   DOI
12 Kushagra Singhal, Jocelyne Vreede, Alireza Mashaghi, Sander J. Tans, Peter G. Bolhuis, "The Trigger Factor Chaperone Encapsulates and Stabilizes Partial Folds of Substrate Proteins", PLoS Comput Biol 11(10): e1004444. (2015). DOI: https://doi.org/10.1371/journal.pcbi.1004444   DOI
13 Kumar, V., Abbas, A. K., Fausto, N., Robbins, S. L. and Cotran, R. S., "Robbins and Cotran Pathologic Basis of Disease (7th ed.)", Elsevier Saunders, St. Louis, Mo (2005).
14 Sung, K., & Kim, I. H. "Expression and purification of recombinant human epidermal growth factor using fusion partners in Escherichia coli", Korean Chemical Engineering Research, 56(5), 711-717. (2018). DOI: https://doi.org/10.9713/kcer.2018.56.5.711   DOI
15 Kim, B.-L., Baek, J. E., Lee, E. G., Lee, H.-W., Kim, C. S., Ahn, J.-O., Lee, H.-W., Kim, I.-H., & Jung, J.-K. "Study on Soluble Expression of Recombinant Human Epidermal Growth Factor Using Various Fusion Partners in Escherichia coli", TT-. Korean Society for Biotechnology, 35. (2007). https://www.earticle.net/Article/A100302
16 Parthasarathy Seshacharyulu, Moorthy P Ponnusamy, Dhanya Haridas, Maneesh Jain, Apar K Ganti & Surinder K Batra, "Targeting the EGFR signaling pathway in cancer therapy, Expert Opinion on Therapeutic Targets", 16:1, 15-31, (2012). DOI: https://doi.org/10.1517/14728222.2011.648617   DOI
17 Carpenter G, Cohen S. "Epidermal growth factor", Tanpakushitsu Kakusan Koso. Protein, Nucleic Acid, Enzyme, 45(13 Suppl), 2116-2122. (2000). DOI: https://doi.org/10.5578/kvj.24183
18 Hyun Sook Kim, Ki Mun Kang, Sang-wook Lee, Jae Boem Na, and Gyu Young Chai, "Effect of Recombinant Epidermal Growth Factors on Irradiated Fibroblast Proliferation", Korean Journal of Radiation Oncology, 24(3), 58. (2006).
19 Ma, Y., Yu, J., Lin, J., Wu, S., Li, S., & Wang, J. "High Efficient Expression, Purification, and Functional Characterization of Native Human Epidermal Growth Factor in Escherichia coli", BioMed Research International, (2016). DOI: https://doi.org/10.1155/2016/3758941
20 Ferrer Soler, L., Cedano, J., Querol, E., & de Llorens, R. "Cloning, expression and purification of human epidermal growth factor using different expression systems Journal of Chromatography", B, Analytical Technologies in the Biomedical and Life Sciences, 788(1), 113-123. (2003). DOI: https://doi.org/10.1016/s1570-0232(02)01035-8   DOI
21 Kang, J. S., La, H. N., Bak, S. U., Eom, H. J., Lee, B. K., & Shin, H. J. "Development of Dermal Transduction Epidermal Growth Factor (EGF) Using A Skin Penetrating Functional Peptide", J. Soc. Cosmet. Sci. Korea, 45(2), 175-184. (2019).   DOI
22 Kim, K. H., Lee, S., & Park, H.-S. "Inhibitory Effects of Three Dimensional Adipose Tissue-Derived Mesenchymal Stem Cell Conditioned Medium on Immune Response and Efficacy Evaluation of its Cream", Asian Journal of Beauty and Cosmetology, 17(1), 25-36. (2019). DOI: https://doi.org/10.20402/ajbc.2018.0255   DOI
23 Woo, G. W. "Recombinant Human Epidermal Growth Factor", Journal of Korean Burn Society, 11(2), 79-84. (2008).
24 Chen, G. F. T., & Inouye, M. "Role of the AGA/AGG codons, the rarest codons in global gene expression in Escherichia coli", Genes and Development, 8(21), 2641-2652. (1994). DOI: https://doi.org/10.1101/gad.8.21.2641   DOI
25 Jung Woo Yi, Hee Jun Kim, Seong Wan Cho, J. S. P. and Y. W. C. "Topical Gel Formulations of Epidermal Growth Factor and Their Wound Healing Effects", Yakhak Hoeji, 40(4), 411-417. (1996).
26 Kim, J.-S., Jang, S.-W., Park, J.-B., Kwon, D.-H., Chang, Y.-J., Jung, H.-M., Han, S.-I., Hong, E.-K., & Ha, S.-J. "Production of Human Interferon ${\beta}$ by Recombinant E. coli Using the Codon Optimized Gene", KSBB Journal, 32, 16-21. (2017). DOI: https://doi.org/10.7841/ksbbj.2017.32.1.16   DOI
27 Tokuoka, M., Tanaka, M., Ono, K., Takagi, S., Shintani, T., & Gomi, K. "Codon optimization increases steady-state mRNA levels in Aspergillus oryzae heterologous gene expression", Applied and Environmental Microbiology, 74(21), 6538-6546. (2008). DOI: https://doi.org/10.1128/AEM.01354-08   DOI
28 Ikemura, T. "Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes: A proposal for a synonymous codon choice that is optimal for the E. coli translational system", Journal of Molecular Biology, 151(3), 389-409. (1981). DOI: https://doi.org/10.1016/0022-2836(81)90003-6   DOI
29 Kane, J. F., Violand, B. N., Curran, D. F., Staten, N. R., Duffin, K. L., & Bogosian, G. "Novel in-frame two codon translational hop during synthesis of bovine placental lactogen in a recombinant strain of Escherichia coli", Nucleic Acids Research, 20(24), 6707-6712. (1992). DOI: https://doi.org/10.1093/nar/20.24.6707   DOI
30 Singh, S. M., & Panda, A. K. "Solubihzation and refolding of bacterial inclusion body proteins", Journal of Bioscience and Bioengineering, 99(4), 303-310. (2005). DOI: https://doi.org/10.1263/jbb.99.303   DOI