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18.97.14.91
18.97.14.91
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SCIE SCOPUS
단일항체 칼럼과 고압 액체크로마토그래피에 의한 유전자 조작 사람 알파 인터페론의 분리 및 정제
Isolation and purification of human interferon - αfrom Recombinant E . coli by Monoclonal Antibody Column and HPLC Gel Filtration
박희섭 , 정미영 , 오명석 , 고중환 , 김현수 , 현형환 ( H . S . Park , M . Y . Jung , M . S . Oh , J . H . Koh , H . S . Kim , H . H . Hyun )
BMB Reports vol. 20 iss. 4 336-344(9pages)
UCI I410-ECN-0102-2008-470-002212062

Immunoadsorbent chromatography technique was applied for the efficient purification of Interferon (rIFN-α) from recombinant E. coli. Monoclonal antibody to rIFN-α was purified using protein A-Sepharose CL-4B chromatography from ascites fluid of BALB/c mice which were innoculated with monoclonal hybridoma cell. The purified antibody was coupled to Affigel-10, and used as an immunoadsorbent for IFN. The recombinant E. coli cells were sedimented and cell-opening was done using 6 M guanidine-HCl. It was loaded to the above immunoadsorbent column and then the bound IFN was eluted from the column with 0.2 M acetic acid. As a result of purification, the specific activity of the purified IFN was 2 × 10^8 IU/㎎ protein and the purification fold was 4100. The amino-terminal sequence of purified IFN was exactly identical to that of natural human leukocyte IFN upto 30 amino acid residues except the lst and 29th unidentified amino acid residues. The molecular weight of the purified IFN was estimated to be 18,600 by SDS-PAGE. Since IFN purified by monoclonal antibody column contains IFN dimer up to 15% further purification was done by HPLC gel filtration. The purity of finally-purified IFN monomer was estimated to be more than 99.9% by SDS-PAGE under non-reducing condition. Finally-purified IFN monomer has several pI points (6.12, 6.06, 5.85, 5.58, and 5.30).

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