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박희섭,정미영,오명석,고중환,김현수,현형환,Park, H.S.,Jung, M.Y.,Oh, M.S.,Koh, J.H.,Kim, H.S.,Hyun, H.H. 생화학분자생물학회 1987 한국생화학회지 Vol.20 No.4
유전자 조작된 대장균이 생산하는 알파 인터페론을 단일 항체 칼럼을 사용하여 효과적으로 정제하였다. rIFN-$\alpha$에 대한 항체를 생산하는 단일클론 융합세 포를 쥐(BALB/c)의 복강에 접종하여 얻은 복강액으로부터 Protein A column을 사용하여 항체를 정제하였다. 이 정제된 항체를 Affigel-10에 결합시켜 단일항체 칼럼을 만든 후, 유전자 조각 장균을 6M 구아니딘 (guanidine-HCl)으로 처리 하여 얻은 인터페론 포함 상등액을 통과시켜 인터페론을 정제하였다. 정제된 인터페론 $2{\times}10^8\;IU/mg$ protein의 specific activity를 가졌으며 분자량은 SDS-PAGE에서 18,600이었고 30개까지 이이노산 서열을 분석한 결과 천연의 human leukocyte IFN-$\alpha$의 것과 동일하였다. 비환원 조건에서 SDS-PAGE 한 결과 약 15%의 인터페론 이중체 (Interferon dimer)를 포함 하고 있었다. 이를 제거하기 위해 생리식염수 용액으로 사용하여 HPLC 젤여과를 행하였다. 최종 정제된 인터페론을 비환원 조건에서 SDS-PAGE 한 결과 인터페론 단일체의 순도는 99.9% 이상인 것으로 판명되었으며, 최종 정제된 인터페론은 약 5개의 등전 전위점을 갖고 있다 (pI = 6.12, 6.06, 5.85, 5.58, 5.30). Immunoadsorbent chromatography technique was applied for the efficient purification of Interferon (rIFN-$\alpha$) from recombinant E. coli. Monoclonal antibody to rIFN-$\alpha$ 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 immunoadsorhent 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 $2{\times}10^8\;IU/mg$ 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 1st 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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단일항체 칼럼과 고압 액체크로마토그래피에 의한 유전자 조작 사람 알파 인터페론의 분리 및 정제
박희섭,정미영,오명석,고중환,김현수,현형환 ( H . S . Park,M . Y . Jung,M . S . Oh,J . H . Koh,H . S . Kim,H . H . Hyun ) 생화학분자생물학회 1987 BMB Reports Vol.20 No.4
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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