Journal of the Korea Academia-Industrial cooperation Society (한국산학기술학회논문지)

The Korea Academia-Industrial cooperation Society (한국산학기술학회)
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Expression of Recombinant Human Epidermal Growth Factor as a Active Form through Codon Optimization with E. coli and Co-expression of Chaperone

코돈 최적화 및 샤페론 공발현을 통한 활성 형태의 재조합 인간 상피세포성장인자의 발현

  • 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)
  • 장은빈 (건양대학교 의료신소재학과) ;
  • 김준수 (건양대학교 의료신소재학과) ;
  • 이우일 (건양대학교 의료신소재학과)
  • Received : 2020.07.09
  • Accepted : 2020.09.04
  • Published : 2020.09.30
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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.

세포 분열 및 성장 촉진에 영향을 주는 상피세포 성장인자(Epidermal Growth Factor, EGF)는 다양한 의학적 용도를 갖고 있는 호르몬 단백질이다. 본 연구에서는 human EGF 유전자를 대장균 코돈에 최적화 하고 pRSET 벡터에 클로닝하여 발현벡터를 구축하였다. Human EGF를 봉입체가 아닌 활성이 있는 형태로의 과량 발현을 위해 코돈의 최적화와 더불어 최초로 샤페론 공발현이 시도되었다. 발현된 Native protein 형태의 재조합 human EGF는 고순도로 정제하기 위해 Ion Exchange Chromatography를 2번 연속적으로 수행하여 순수 분리 정제되었고, ELISA 분석결과 99% 이상으로 재조합 EGF의 활성도가 상업용 EGF와 유사하게 나타났으며, 세포증식시험 결과 인간 재조합 EGF는 인체 피부 섬유아세포의 세포증식을 촉진하는 것으로 확인 되었다. 본 연구의 인간 EGF 발현 시스템은 양적인 측면 뿐 아니라 성공적인 활성형태의 발현으로 추가적인 재접힘 과정 및 N 말단의 융합부분을 제거하기 위한 크로마토그래피 작업이 필요가 없다는 점에서 기존의 방법들에 대체 될 수 있는 효과적인 인간 EGF 발현 시스템을 제공하고 있다.

Keywords

References

  1. 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
  2. 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
  3. 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
  4. 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
  5. 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).
  6. 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
  7. 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
  8. 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).
  9. 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). https://doi.org/10.15230/SCSK.2019.45.2.175
  10. 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
  11. Woo, G. W. "Recombinant Human Epidermal Growth Factor", Journal of Korean Burn Society, 11(2), 79-84. (2008).
  12. 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).
  13. 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
  14. 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
  15. 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
  16. 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
  17. 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
  18. 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
  19. 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
  20. 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)
  21. 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)
  22. Hartl, F. U. "Molecular chaperones in cellular protein folding", Nature, 381(6583), 571-580. (1996). DOI: https://doi.org/10.1038/381571a0
  23. 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
  24. 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
  25. 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
  26. 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).
  27. Suk Young Kim, "The Role of Heat Shock Protein in Perinatal Fields", Korean J Perinatol Vol. 16, No. 1(2005).
  28. 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
  29. 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
  30. 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