Korean Chemical Engineering Research

The Korean Institute of Chemical Engineers (한국화학공학회)
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Expression and Purification of Recombinant Human Epidermal Growth Factor Using Fusion Partners in Escherichia coli

융합 파트너를 이용한 인간 상피세포성장인자의 재조합 대장균에서 발현과 정제 연구

  • Sung, Keehyun (Department of Chemical Engineering and Applied Chemistry, Chungnam National University) ;
  • Kim, In Ho (Department of Chemical Engineering and Applied Chemistry, Chungnam National University)
  • 성기현 (충남대학교 응용화학공학과) ;
  • 김인호 (충남대학교 응용화학공학과)
  • Received : 2018.07.04
  • Accepted : 2018.08.14
  • Published : 2018.10.01
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Abstract

Human epidermal growth factor (hEGF) can stimulate the division of various cell types and has potential clinical applications. Since the protein contains three intra-molecular disulfide bonds, the high expression of active hEGF in Escherichia coli has not been well researched, We fused the hEGF gene with a small ubiquitin-related modifier gene (SUMO) by synthesizing an artificial SUMO-hEGF fusion gene that was highly expressed in E. coli (DE3) strain. The optimal expression level of the soluble fusion protein, SUMO-hEGF with IPTG (Isopropyl-${\beta}$-D-Thiogalactopyranoside), was up to 38.9% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography and cleaved by a SUMO-specific protease to obtain the native hEGF, which was further purified by Ni-NTA affinity chromatography. The result of the reverse-phase HPLC showed that the purity of the recombinant cleaved hEGF was greater than 98%.

상피세포 성장인자(Epidermal Growth Factor, EGF)는 세포 분열을 자극하고 의약적 용도가 다양하다. EGF는 3개의 이황화 결합을 갖고 불용성으로, 대장균에서 고효율 발현에 대한 연구가 잘 이루어지지 않았다. EGF 유전자를 작은 유비퀴틴 관련 유전자(small ubiquitin-related modifier gene, SUMO)와 결합하고 DE3 대장균에서 발현시켰다. IPTG (Isopropyl-${\beta}$-D-Thiogalactopyranoside)로 유도하여 대장균 세포 단백질의 38.9%로 융합단백질이 발현되었고, Ni-NTA 친화성 크로마토그래피로 분리하였다. 그 후 유비퀴틴 분해효소반응으로 융합단백질에서 EGF를 얻은 후 다시 Ni-NTA 크로마토그래피로 분리 하였다. 최종적으로 정제된 EGF의 순도는 HPLC로 분석하였으며, 98%이상의 순도를 얻을 수 있었다.

Keywords

References

  1. Kim, B.-L., Baek, J. E., Kim, C. S., Lee H.-W., Ahn, J. O., Lee, H. W., Jung, J.-K., Lee, E. G. and Kim, I. H., "Study of Soluble Expression of Recombinant Human Epidermal Growth Factor using Various Fusion Partners in E. coli," KSBB Journal, 23(3), 205-212(2008).
  2. Ferrer, S., Cedano, J., Querol, E. and de Llorens, R., "Cloning, Expression and Purification of Human Epidermal Growth Factor using Different Expression Systems," J. Chromatogr B Analyt Technol Biomed Life Sci., 788(1), 113-23(2003). https://doi.org/10.1016/S1570-0232(02)01035-8
  3. 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).
  4. Ma, Y., Yu, J., Lin, J., Wu, S., Li, S. and Wang, J., "High Efficient Expression, "Purification, and Functional Characterization of Native Human Epidermal Growth Factor in Escherichia coli," Biomed Res Int., 3758941, Epub (2016).
  5. Hollenberg, M. D. and Gregory, H., "Epidermal Growth Factor-Urogastrone: Biological Activity and Receptor Binding of Derivatives," Molecular Pharmacology, 17(3), 314-320(1980).
  6. Zhou, Y., Wu, Q., Wu, Z., Zheng, X., Ding, C., Li, X. and Su, Z. "High-level Expression and Purification of Human Epidermal Growth Factor with SUMO Fusion in Escherichia coli.," Protein Pept Lett., 13(8), 785-92(2006). https://doi.org/10.2174/092986606777841280
  7. Sung, K. H. and Kim, I. H., "Development of Purification Process of Recombinant Human Vascular Endotherial Growth Factor (VEGF) using Fusion Protein," Korean Chemical Engineering Research, 55(3), 369-378(2017). https://doi.org/10.9713/KCER.2017.55.3.369
  8. Herbst, R. S., "Review of Epidermal Growth Factor Receptor Biology," International Journal of Radiation Oncology, Biology, Physics, 59(2 Suppl), 21-26(2004). https://doi.org/10.1016/j.ijrobp.2003.10.027
  9. Kim, J. H., Hwang, W. S. and Kim, I. H., "Simple Preparation of Immobilized-metal Affinity Chromatography Media," Korean Journal of Chemical Engineering, 26(6), 1693-1695(2009). https://doi.org/10.1007/s11814-009-0276-6