KSBB Journal

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
권호 표지

Study on the soluble exoression of recombinant human eoidermal growth factor using various fusion oartners in Escherichia coli

재조합 대장균에서 다양한 융합 파트너를 이용한 인간 상피세포성장인자의 발현 연구

  • Kim, Byung-Lip (Department of chemical Engineering, Chungnam National University) ;
  • Baek, Jung-Eun (Division of Biotechnology R&BD, Korea Research Institute of Bioscience and Biotechnology) ;
  • Kim, Chun-Sug (Division of Biotechnology R&BD, Korea Research Institute of Bioscience and Biotechnology) ;
  • Lee, Hyeok-Weon (Division of Biotechnology R&BD, Korea Research Institute of Bioscience and Biotechnology) ;
  • Ahn, Jung-Oh (Division of Biotechnology R&BD, Korea Research Institute of Bioscience and Biotechnology) ;
  • Lee, Hong-Weon (Division of Biotechnology R&BD, Korea Research Institute of Bioscience and Biotechnology) ;
  • Jung, Joon-Ki (Division of Biotechnology R&BD, Korea Research Institute of Bioscience and Biotechnology) ;
  • Lee, Eun-Gyo (Division of Biotechnology R&BD, Korea Research Institute of Bioscience and Biotechnology) ;
  • Kim, In-Ho (Department of chemical Engineering, Chungnam National University)
  • 김병립 (충남대학교 화학공학과) ;
  • 백정은 (한국생명공학연구원 바이오의약산업화실) ;
  • 김천석 (한국생명공학연구원 바이오의약산업화실) ;
  • 이혁원 (한국생명공학연구원 바이오의약산업화실) ;
  • 안정오 (한국생명공학연구원 바이오의약산업화실) ;
  • 이홍원 (한국생명공학연구원 바이오의약산업화실) ;
  • 정준기 (한국생명공학연구원 바이오의약산업화실) ;
  • 이은교 (한국생명공학연구원 바이오의약산업화실) ;
  • 김인호 (충남대학교 화학공학과)
  • Published : 2008.06.30
Download PDF
⟨ Previous Next ⟩

Abstract

The efficient soluble expression of human epidermal growth factor (hEGF) was achieved by using functional fusion partners in cytoplasm and periplasm of Escherichia coli (E. coli). hEGF was over-expressed in inactive inclusion body form in cytoplasm of E. coli due to improper disulfide bond formation and hydrophobic interaction, yielding about 5.9 mg/L in flask culture. Six functional fusion partners were introduced by linking to N-terminal part of hEGF gene for the high-level expression of soluble and active hEGF in cytoplasm and peri plasm region. Three fusion partners for cytoplasmic expression such as acidic tail of synuclein (ATS), thioredoxin (Trx) and lipase, and three fusion partners for periplasmic expression such as periplasmic cystein oxidoreductases (DsbA and DsbC) and maltose binding protein (MBP) were investigated. hEGF fused with ATS and DsbA showed over 90% of solubility in cytoplasm and periplasm, respectively. Especially DsbA was found to be an efficient fusion partner for soluble and high-level expression of hEGF, yielding about 18.1 mg/L and three-fold higher level compared to that of insoluble non-fusion hEGF in cytoplasm. Thus, heterologous proteins containing complex disulfide bond and many hydrophobic amino acids can effectively be produced as an active form in E. coli by introducing a suitable peptide or protein.

본 연구의 목적은 위치특이적 펩타이드 및 단백질을 사용하여 재조합 대장균에서 활성형 인간 상피세포성장인자(hEGF)를 고효율로 발현할 수 있는 방법을 찾아내는 데 있다. 재조합 대장균내 cytoplasm 및 periplasm 영역에서 hEGF의 발현을 위해 각각 세개의 응합 펩타이드 및 단백질을 선정하여 상호 비교하였다. 재조합 대장균에서 hEGF의 발현유도시 대부분 불용성 단백질로 생산되는 현상을 극복하기 위해 cytoplasm영역에서는 ATS, thioredoxin, 리파제를 융합파트너로 사용하였으며 periplasm 영역에서는 foldase인 DsbA와 DsbC, 용융성 고발현 단백질인 maltose binding protein을 선택하여 사용하였다. Periplasm영역에서 발현유도를 시키는 융합단백질의 경우 cytoplasm영역에서의 발현양도 용융성 형태로 고발현 되는 것을 알 수 있었으며 전체적으로 약 2배가량의 용융성 형태로 발현되었다. hECF의 발현율을 가장 높일 수 있는 융합단백 질은 maltose binding protein이었으나 발현된 융합단백질의 24%가 불용성 단백질로 형성되어 활성형 형태로 얻는 데 한계가 있었으며, 활성형 형태로 hEGF의 발현을 위해서는 DsbA를 응합단백질로 사용한 경우에 18.1 mg/L로 가장 높은 발현농도를 보였다. Cytoplasm 영역에서 발현유도를 한 경우에는 ATS와 thioredoxin을 응합파트너로 hEGF를 발현한 경우 용융성 형태로 높은 발현율을 보였다. 특히 ATS와 같은 펩타이드를 N-말단에 융합시킨 경우 불용성을 방지하는 효과를 보여 이황화결합의 불완전성이나 소수성으로 인해 불용성 단백질로 발현되는 기존의 단백질을 활성형 형태로 발현하는데 될 수 있음을 확인할 수 있었다.

Keywords

References

  1. Cohen, S. (1962), Isolation of a mouse submaxillary gland protein accelerationg incisor eruption and eyelid opening in the new-born animal. J. Biol Chem. 237, 1555-1562
  2. Savage, C. R. and C. Cohen (1972), Epidermal growth factor and a new derivative. J. Biol Chem. 247, 7609-7611
  3. Cohen, S., G. Capenter, and K. J. Lembach (1975), Interaction of epidermal growth factor (EGF) with cultured fibroblasts. Adv. Metab. Disord. 8, 265-284
  4. Gregory, H. and I. R. Willshire (1975), The isolation of the urogastrones-inhibitors of gastric acid secretion from human urine. Hoppe-Seyler's Z. Physiol. Chem. 356, 1765-1774 https://doi.org/10.1515/bchm2.1975.356.2.1765
  5. Cohen, S., Mitchell, W. M., and Talylor, J. M. (1972), Epidermal growth factor (EGF) -physical and chemical properties. J. Biol Chem. 247, 5928-5934
  6. Bhall, N. P., K. Patel, and R. T. Borchardt (1990), Chemical pathways of peptide degration. I. Oeamidation of adrenocotropic hormone. Pharrn. Res. 7, 593-597 https://doi.org/10.1023/A:1015862026539
  7. Cuatreeasas, P. (1972), Isolation of the insulin receptor of liver and fat cell membranes. Proc. Nat. Acad. Sci. 69, 318-322
  8. Hasegawa, N. and Yamamoto, K. (1992), Epidermal growth factor supresses in vitro senescence in the ability of human unbilical vein endothelial cells to proliferate, but not in the ability to produce prostacyclin. Mech. Agei. Develop. 66, 107-114 https://doi.org/10.1016/0047-6374(92)90077-Q
  9. aka, T., Sakamoto, S., Miyoshi, K. I., Fuwa. T., Yoda. K., Yamasaki. M., Tamura. G., and Miyake. T. (1985), Synthesis and secretion of human epidermal growth factor by Escherichia coli. Proc. Nat. Acad. Sci. 82, 7212-7216
  10. Yamagata, H., K. Nakahama, Y. Suzuki, A. Kakinuma, N. Tsukagoshi, and S. Udaka (1989), Use of Badllus brevis for efficient synthesis and secretion of human epidermal growth factor. Proc. Natl. Acad Sci. USA 86, 3589-3591
  11. A. Miyauchi, S. Ebisu, K Uchida, M. Yoshida, M. Ozawa, T. Tojo, K. Kadowaki, and H. Takagi (1998), Pilot scale production of a recombinant human epidermal growth factor, secreted by Bacillus brevis, using expanded bed adsorption. J. Int. Micro. Biol. 21, 208-214
  12. Udaka, S., Tsukagoshi, N., and Yamagata, H. (1989), Bacillus brevis, a host bacterium for efficient production of useful proteins. Biotechnol. Gent. Eng. Rev. 7, 113-146 https://doi.org/10.1080/02648725.1989.10647857
  13. M. Date, H. Itaya, H. Matsui, and Y. Kikuchi (2006), Secretion of human epidermal growth factor by Corynebacterium glutamicum. Applied Microbiology 42, 66-70 https://doi.org/10.1111/j.1472-765X.2005.01802.x
  14. Haiyi Zhang, Zhimin Li, Yue Qian, Qian Zhang, Peng Du, Renbao Gan, and Qin Ye (2006), Cultivation of recombinant Escherichia coli for secretory production of human epidermal growth factor under control of $P_L$ promoter. EMT. 10, 1-8
  15. Puthalakath V. Hamsa, Pradeep Kachroo, and Bharat B. Chatto (1998), Production and secretion of biologically active human epidermal growth factor in Yarrowia lipolytica Curr. Genet. 33, 231-237
  16. H. Moon, H. Kim, S. K. Rhee, E. S. Choi, I. H. Kim, and S. I. Hong (2002), Optimal strategy of pH control in the production of recombinant human epidennal growth factor by Hansenula polymorpha. Proc. Biochem 38, 487-495 https://doi.org/10.1016/S0032-9592(02)00172-3
  17. 허일섭, 박호군, 김희철, 오용익, 이사기, 최의성, 손정훈 (1994), 인체표피성장인자의 유전자를 갖는 벡터 및 그를 이용한 인체표피성장인자의 생산방법. 대한민국특허. 출원번호 10-1994-0019637
  18. Siegal, C. B., R. P. Nordan, D. J. Fitzgerald, and I. Pastan (1990), Cell-specific toxicity of a chimeric protein compsed of interleukin-6 and Psudomonas exotoxin (lL6-PE40) on tumor cells. Mol. Cell BioI. 10, 2443-2447 https://doi.org/10.1128/MCB.10.6.2443
  19. S. Jana and J. K. Deb (2005), Strategies for efficient production of heterologous proteins in Escherichia coli. Appl. Mcrobiol Biotech 67, 289-298 https://doi.org/10.1007/s00253-004-1814-0
  20. Takanorri aka, Shunji Sakamoto, Ken-ichi Miyoshi, Tom Fuwa, Koji Yoda, Makari Yamasaki, Gaku Tamura, and Tetsuo Miyake (1985), Synthesis and secretion of human epidermal growth factor by. Escherichia coli. Proc. Natl. Acad. Sci. USA 82, 7212-7216
  21. Kazuko Morioka-Fujimoto, Ryuji Mammoto, and Tsunehiko Fukuda (1991), Modified enterotoxin signal sequences increase secretion level of the recombinant human epidermal growth factor in Escherichia coli. J. BioI. Chem. 266, 728-1732
  22. Nobuki Hayase, Akira Ishiyama, and Mitsum Niwano (1994), Secretion of human epidermal growth factor (hEGF) in autotrophic culture by a recombinant hydrogen-utilizing bacterium Pseudomonas pseudoflava, carrying broad-hast-range EGF secretion vector pKSEGF2 Applied Env. Micro 60, 3336-3342
  23. S. Sivakesava, Z. N. Xu, Y. H. Chen, J. Hackett, R. C. Huang, E. Lam, T. L. Lam, K. L. Siu, R. S. C. Wong, and W. K. R. Wong (1999), Production of excreted human epidermal growth factor (hEGF) by an efficient recombinant Escherichia coli. Proc. Biotech. 34, 893-900
  24. Mina Ebrahirni-Rad, Morteza Azarnoosh, Aftab Bashir, and Vladimir V. Bakayev (2004), Novel vector for expression/secretion of properly folded eukaryotic proteins: a comparative study on cytoplasmic and periplarnic expression of human epidermal growth factor (hEGF) in Escherichia coli. Iranian Biomedical Journal. 2, 51-61
  25. Yi-Lin Liu, Liang-Min Huang, Wen-Po Lin, Chung-Chin Tsai, Tsung-Shull Lin, Yu-Hua Hu, H. Shin Chen, Jun-Ming Han, Hsian-Jenn Wang, and Yu-Tien Liu (2006), Secretion of biologically active hEGF from Escherichia coli using Yersinia pestis Cafl signal peptide. J. Microbiol Immunol Infect. 39, 366-371
  26. 박세철, 남정현, 김정근, 권태종, 고인영, 유광현 (1996), PelB Signal Sequence로 유도된 제조합 인간 상피세포 증식인자 분비발현 벡터의 제조. 한국미생물공학회 24, 553-559