Korean Chemical Engineering Research

  • 제55권3호
  • /
  • Pages.369-378
  • /
  • 2017
  • /
  • 0304-128X(pISSN)
  • /
  • 2233-9558(eISSN)
한국화학공학회 (The Korean Institute of Chemical Engineers)
권호 표지
DOI QR코드

DOI QR Code

융합 단백질을 이용한 재조합 인간 혈관내피세포 성장인자의 정제공정 개발

Development of Purification Process of Recombinant Human Vascular Endotherial Growth Factor (VEGF) using Fusion Protein

  • 성기현 (충남대학교 응용화학공학과) ;
  • 김인호 (충남대학교 응용화학공학과)
  • 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)
  • 투고 : 2017.01.16
  • 심사 : 2017.03.21
  • 발행 : 2017.06.01
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

혈관 내피세포 성장인자(Vascular endotherial growth factor, VEGF)는 혈관 투과율 조절이나 혈관 생성에 관련된 단백질로 임상용으로 쓰일 가능성이 높다. 이 단백질은 고순도와 고효율로 상업적으로 대량생산이 필요하다. 유비퀴틴 융합 단백질로 온화한 조건에서 용해시키기 위해 다양한 조건을 연구하였고, pH와 변성제 변화를 시도하였다. BL21 (DE3) 대장균 숙주세포에서 pET28-a 벡터를 사용하여 재조합 대장균을 제조하여, 20 L의 회분식 배양으로 14 g/L농도의 세포배양을 하였다. 발효 후UBP1 효소 분해와 재접힘 단계를 포함한 4단계의 크로마토그래피 공정으로 구성된 정제공정으로 VEGF를 정제하였다. 유비퀴틴 융합단백질로 2 M 요소와 pH10 온화한 조건에서 VEGF의 정제가 가능하였다. 2번의 Ni-affinity 크로마토그래피컬럼을 이용하여 고효율의 재접힘과 이합체화 공정을 수행하였다. DEAE (Diethyl Amino Ethyl) 음이온 교환 컬럼을 통하여 변형체(multimeric, misfolded)단백질과 endotoxin을 제거 할 수 있었다. 젤 여과 크로마토그래피를 이용하여 dimer와 monomer를 분리 하여 이합체화 VEGF를 제조하였다. 최종 VEGF의 특성분석을 SDS-PAGE (Soidum Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) 전기영동, RP-HPLC (Reversed Phase High Performance Liquid Chromatography)으로 하여 순도 97% (RP-HPLC기준)를 얻었다.

Vascular endotherial growth factor (VEGF) is a potent mitogen that stimulates vascular permeability and angiogenesis and has a potential in therapeutic applications. An industrial production method that provides high yield as well as purity is needed. Researches for various factors of mild solubilization with combination of ubiquitin fusion protein to increase solubility were carried out as well as by changing pH and denaturant concentration. Usage of pET28-a bacteral expression vector in BL21 (DE3) host cell was capable of producing approximately 14 g/L VEGF fusion protein in 20L fermentor. A purification process consisting of four chromatography steps including refolding and digestion with UBP1 resulted in mild solublization under the conditions of 2M urea and pH 10.0 due to ubiquitin fusion tag protein that increases in solubility of target protein VEGF. High yield of refolding and dimerization could be obtained between two step Ni-affinity chromatography. Multimeric and misfolded proteins and endotoxin were removed by DEAE anion exchange chromatography. Final monomers were removed from dimers by gel filtration chromatography. Characterization analysis of purified dimeric VEGF was performed using SDS-PAGE and RP-HPLC with a purity of 97%.

키워드

참고문헌

  1. Abe, T., Okamura, K., Ono, M., Mori, T., Hori, S. and Kuwano, M., "Induction of Vascular Endothelial Tubular Morphogenesis by Human Glioma Cells : a Model System for Tumor Angiogenesis," J. Clin. Invest., 92, 54-61(1993). https://doi.org/10.1172/JCI116599
  2. Ferrara, N., "Role of Vascular Endothelial Growth Factor in the Regulation of Angiogenesis," Kidney Int., 56, 794-814(1999). https://doi.org/10.1046/j.1523-1755.1999.00610.x
  3. Ferrara, N., "Molecular and Biological Properties of Vascular Endothelial Growth Factor," J. Mol. Med., 77, 527-543(1999). https://doi.org/10.1007/s001099900019
  4. Drake, C. J., LaRue, A., Ferrara, N. and Little, C. D., "VEGF Regulates Cell Behavior during Vasculogenesis," Dev. Biol., 224, 178-188(2000). https://doi.org/10.1006/dbio.2000.9744
  5. Nissen, N. N., Polverini, P. J., Koch, A. E., Volin, M.V., Gamelli, R. L. and DiPietro, L. A., "Vascular Endothelial Growth Factor mediates Angiogenic Activity during the Proliferative Phase of Wound Healing," Am.J. Pathol., 152, 1445-1452(1998).
  6. Ferrara, N., "Vascular Endothelial Growth Factor : Basic Science and Clinical Progress," Endocrine Reviews., 25, 581-611(2004). https://doi.org/10.1210/er.2003-0027
  7. Houck, K. A., Ferrara, N., Winer, J., Cachianes, G., Li, B. and Leung, D. W., "The Vascular Endothelial Growth Factor Family: Identification of a Fourth Molecular Species and Characterization of Alternative Splicing of RNA," Mol. Endocrinol., 5, 1806-1814(1991). https://doi.org/10.1210/mend-5-12-1806
  8. Xie, K., Wei, D., Shi, Q. and Huangb, S., "Constitutive and Inducible Expression and Regulation of Vascular Endothelial Growth Factor," Cytokine & Growth Factor Reviews., 15, 297-24(2004). https://doi.org/10.1016/j.cytogfr.2004.04.003
  9. Lee, S.-B., Park, J. S., Lee, S., Park, J., Yu, S. Kim, D., Kim, H, Byun, T. H., Baek, K., Ahn, Y.-J. and Yoon, J., "Overproduction of Recombinant Human VEGF (Vascular Endothelial Growth Factor) in Chinese Hamster Ovary Cells," J. Microbiol. Biotechnol., 18, 183-87(2008).
  10. Wiesmann, C., Fuh, G., Christinger, H. W. Eigenbrot, C., Wells, J. A. and de Vos, A. M., "Crystal Structure at 1.7 A Resolution of VEGF in Complex with Domain 2 of the Flt-1 Receptor," Cell, 91, 695-704(1997). https://doi.org/10.1016/S0092-8674(00)80456-0
  11. Keck, R. G., Berleau, L., Harris, R. and Keyt, B. A., "Disulfide Structure of the Heparin Binding Domain in Vascular Endothelial Growth Factor: Characterization of Posttranslational Modifications in VEGF," Arch. Biochem. Biophys., 344, 103-113(1997). https://doi.org/10.1006/abbi.1997.0145
  12. Pizarro, S. A., Gunson, J., Field, M. J., Dinges, R., Khoo, S., Dalal, M., Lee, M., Kaleas, K. A., Moiseff, K., Garnick, S., Reilly, D. E., Laird, M. W. and Schmelzer, C. H., "High-yield Expression of Human Vascular Endothelial Growth Factor VEGF (165) in Escherichia coli and Purification for the Therapeutic Applications," Protein Expr Purif., 72(2), 184-193(2010). https://doi.org/10.1016/j.pep.2010.03.007
  13. Scrofani, S. D. B., Fabri, L. J., Xu, P. Maccarone, P. and Nash, A. D., "Purification and Refolding of Vascular Endothelial Growth Factor-B," Protein Science, 9(10), 2018-2025(2000). https://doi.org/10.1110/ps.9.10.2018
  14. Lee, I. L., Li, P. S., Yu, W. L. and Shen, H. H., "Prokaryotic Expression, Refolding, and Purification of Functional Human Vascular Endothelial Growth Factor Isoform 165: Purification Procedures and Refolding Conditions Revisited," Protein Expr Purif., 76(1), 54-58(2011). https://doi.org/10.1016/j.pep.2010.08.014
  15. Ko, K. Y. and Kim, I. H., "Moment Analysis (MA) of Lysozyme in Cation Exchange High Performance Liquid Chromatography (HPLC)," Korean Chem. Eng. Res., 54(4), 487-493(2016). https://doi.org/10.9713/kcer.2016.54.4.487