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Received January 16, 2017
Accepted March 21, 2017
articles This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/bync/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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융합 단백질을 이용한 재조합 인간 혈관내피세포 성장인자의 정제공정 개발

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

충남대학교 응용화학공학과, 34134 대전광역시 유성구 대학로 99
Department of Chemical Engineering and Applied Chemistry, Chungnam National University, 99, Daehak-ro, Yuseong-gu, Daejon, 34134, Korea
ihkim@cnu.ac.kr
Korean Chemical Engineering Research, June 2017, 55(3), 369-378(10), 10.9713/kcer.2017.55.3.369 Epub 2 June 2017
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Abstract

혈관 내피세포 성장인자(Vascular endotherial growth factor, VEGF)는 혈관 투과율 조절이나 혈관 성에 관련된 단 백질로 임상용으로 쓰일 가능성이 높다. 이 단백질은 고순도와 고효율로 상업적으로 대량생산이 필요 다. 유비퀴틴 융합 단백질로 온화한 조건에서 용해시키기 위해 다양한 조건을 연구하였고, pH와 변성제 변화를 시 하였다. BL21 (DE3) 대장균 숙주세포에서 pET28-a 벡터를 사용하여 재조합 대장균을 제조하여, 20 L의 회분식 배 으로 14 g/L농도 의 세포배양을 하였다. 발효 후UBP1 효소 분해와 재접힘 단계를 포함한 4단계의 크로마토그래피 공 으로 구성된 정 제공정으로 VEGF를 정제하였다. 유비퀴틴 융합단백질로 2 M 요소와 pH10 온화한 조건에서 VEGF의 정제가 가능하 였다. 2번의 Ni-affinity 크로마토그래피 컬럼을 이용하여 고효율의 재접힘과 이합체화 공정을 수행하 다. 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 VEG 의 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%.

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