Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Partial Purification and Quantification of Insulin-like Growth Factor-I from Red Deer Antler

녹용으로부터 Insulin-like Growth Factor-I의 일부정제 및 정량

  • Gu, Lijuan (Department of Food Science and Technology, College of Agriculture and Biotechnology, Chungnam National University) ;
  • Mo, Eun-Kyoung (Department of Food Science and Technology, College of Agriculture and Biotechnology, Chungnam National University) ;
  • Fang, ZheMing (Department of Food Science and Technology, College of Agriculture and Biotechnology, Chungnam National University) ;
  • Sun, BaiShen (Department of Food Science and Technology, College of Agriculture and Biotechnology, Chungnam National University) ;
  • Zhu, XueMei (Department of Food Science and Technology, College of Agriculture and Biotechnology, Chungnam National University) ;
  • Sung, Chang-Keun (Department of Food Science and Technology, College of Agriculture and Biotechnology, Chungnam National University)
  • ;
  • 모은경 (충남대학교, 농업생명과학대학, 식품공학과) ;
  • ;
  • ;
  • ;
  • 성창근 (충남대학교, 농업생명과학대학, 식품공학과)
  • Published : 2007.10.30
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Abstract

Deer antler tissue contains the most rapidly growing bone in the animal kingdom. Thus, it is likely that growing antler tissue is a rich source of local paracrine bone-stimulating factors. Growth factors, at least the insulin-like growth factor (IGF), control the bone-remodelling process. In this study, we tried to isolate and purify IGF-I from fresh antler tissue by the routine isolation and purification of protein. The purification involved ammonium sulfate precipitation, DEAE-Sepharose CL-60 ion-exchange chromatography, CM-Sepharose CL-6B ion-exchange chromatography, and Sephadex G-50 chromatography. Purified fractions from each step were analyzed by high-performance liquid chromatography (HPLC), SDS polyacrylamide gel electrophoresis (SDS-PACE), Dot-blot, and Western-blot methods. Furthermore, the quantification of partially purified IGF-I was calculated by enzyme-linked immunosorbent assays (ELISA) using antibody to human recombinant IGF-1. SDS-PAGE analysis of the final fraction yielded two molecular bands and the signal band was at 12 kDa on the Western-blot film. This purified IGF-I fraction showed a peak at retention time of eight min. The quantity of IGF-I in 20 g deer antler tissue as starting weight was calculated using a standard curve to be 2910 ng/ml, and total IGF-I amount is 0.291 g. The results show that IGF-I, which can be found in deer antler, can be partially purified and quantified by classic protein isolation methods.

사슴뿔은 동물세계에서 가장 빨리 성장하는 조직이다. 따라서 성장중인 사슴뿔은 뼈 성장을 촉진하는 인자가 풍부하게 포함된 것으로 생각된다. 이들 성장인자들 중 IGF-1은 뼈를 자라게 하는 조골세포의 대사에 중요한 역할을 한다고 알려져 있어 이를 정제하고자 하였다. IGF-1의 정제는 상대라고 불리는 신선한 사슴뿔을 유안침전, DEAE-Sepharose CL-6B 이온교환수지, CM-Sepharose CL-6B 양이온교환수지, Sephadex G-50의 순차적인 방법으로 할 수 있었다. 각 과정마다 IGF-1의 거동을 HPLC, SDS-PAGE, Dot blot, 그리고 western blot으로 분석하였다. IGF-1의 정량은 ELISA기술로 재조합 인간 IGF-1을 이용하여 계산되었으며, 최종 분별 액은 두 개의 단백질을 보였으나, Western-blot에서 작은 분자량인 12 kDa으로 최종 판명할 수 있었다. 정제된 단백질은 HPLC에서 retention 시간 8분만에 검출되었으며, 총 농도는 2910 ng/ml 이고 중량은 0.291 g 이었다.

Keywords

References

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