KSBB Journal

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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Purification of TGF-$\beta$ 1 from Human Platelets by an Improved Method

개량된 방법에 의한 사람혈소판으로부터 TGF-$\beta$ 1의 분리

  • 신충건 (경북대학교 자연과학대학 생화학과) ;
  • 김상국 (경북대학교 자연과학대학 생화학과) ;
  • 문병조 (경북대학교 자연과학대학 생화학과) ;
  • 김평현 (강원대학교 자연과학대학 생명과학부) ;
  • 전계택 (강원대학교 자연과학대학 생명과학부) ;
  • 남상욱 (강원대학교 사범대학 과학교육학부) ;
  • 김장환 (대구효성카톨릭대학교 의과대학 생화학교실) ;
  • 이종원 (대구효성카톨릭대학교 의과대학 생화학교실)
  • Published : 1999.02.01
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Abstract

Transforming growth factor $\beta$1(TGF-$\beta$1) has potentials to be used as a new therapeutic agent. However, studies with TGF-$\beta$ were hindered by its high cost. In this study, we developed an improved method to purify TGF-$\beta$1 from human platelets, for which four purification steps were used: platelet extraction, gel filtration, cation exchange chromatography, and reverse phase high performance liquid chromatography. After a final step of purification, a pure protein with a molecular weight of 25,000 corresponding to the commercially available TGF-$\beta$1 was obtained, which were confirmed by silver staining and Western blotting after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It was confirmed by the inhibitory effects of TGF-$\beta$1 on a mink lung epithelial cell line that the purified TGF-$\beta$1 had its biological activity, whose activity is slightly higher than that of the commercially available TGF-$\beta$1. About 3.7$\mug of the purified TGF-$\beta$1 was obtained from 10 units of concentrated human platelets, the final yield of which is about 21%.

Transforming growth factor $\beta$1(TGF-$\beta$1)은 여러 가지 생물학적 활성을 가지는 관계로 의학적 치료제로서 사용될 가능성이 크다. 본 연구에서는 혈소판추출, 젤여과, 양이온교환 크로마토그래피 및 역상 HPLC등 네 단계의 정제과정으로 이루어져 있는 정제공정을 이용한 TGF-$\beta$1을 값싸고 효울적으로 정제하였다. 이 과정을 거쳐 최종적으로 얻어진 TGF-$\beta$1은 비환원조건하에서 SDS-PAGE를 행한 결과 구매된 TGF-$\beta$1 표준품과 일치한 위치에서 한 개의 band가 관찰되어 순수하다는 것을 확인하였으며 또한 이것이 Westem blot를 통하여 TGF-$\beta$1 항체와 결합하는 것으로부터 TGF-$\beta$1임을 확인하였다 또한, mink lung epithelial cell line 을 이용한 성장저해 실험을 통해 정제된 TGF-$\beta$1이 구매되TGF-$\beta$1 표준품보다 조금 높은 활성을 가지는 것을 확인하였다 최종적으로 농축혈소판 10단위로부터 약 3.7$\mu$g의 정제된 TGF-$\beta$1이 얻어져 그 최종수율은 약 21%였다.

Keywords

References

  1. Growth Factors v.8 Physiological Action and Clinical Applications of TGF-β Roberts, A.B.;Sporn, M.B.
  2. J-Biol-Chem v.271 Requirement of TGF-β thype Ⅱ Receptor for TGF- β -induced Proliferation and Growth Inhibition Zhao Y.;Young S.L.
  3. In-Vitro-Cell-Dev-Biol-Anim v.30A The Effect of TGF- β on Fibroblast Cell Proliferation in Intact Connective Tissue in Vitro Franzen L.;Dahlquist D.
  4. Science v.237 Transforming Growth Factor β In-duces Accelerated Healing of Incisional Wounds in Rats Mustoe T.A.
  5. Arch-Otolaryngol-Head-neck-Surg v.122 TGF β1 Improves Wound Healing and Random Flap Survival in Normal and Irradiated Rats Nall A.V.;Brownlee R.E.;Colvin C.P.;Schultz G.;Fein D.;Cassisi N.J.;Nguyen T.;Kalra A.
  6. Surgery v.113 Acceleration of Tissue Repair by TGF β1: Identification of in vivo Mechnism of Action with Radiotherapy-induced Specific Healing Deficits Cromack D.T.;porras R.S.;Purdy J.A.;pierce G.F.;mustoe T.A.
  7. Growth Factors v.2 Accelerated Healing of Ulcer Wounds in the Rabbit Ear by Recombinant Human Transforming Growth Factor- β1 Beck L.S.;Chen T.L.;Hirabayashi S.E.;Deguzman L.;Lee W.P.;McFatridge L. L.;Xu Y.;Bates R.L.;Ammann A.J.
  8. Science v.226 Growth Inhibitor from BSC-1 Cells Colsely Related to Tpe β Transforming Growth Factor Tucker R.F.;Shipley G.D.;Moses H.L.;Holley R.W.
  9. Cancer Res v.46 Reversible inhibition of normal human prokeratinocyte proliferation by type β transfo-rming growth factor-growht inhibitor in serum-free medium Shipley G.D.;pittelknow M.R.;Wille J.J.J.;Scott R.E.;Moses H.L.
  10. Blood v.72 Transforming Growth Factor β Selectively Inhibits Normal and Leckemic Human Bone Marrow Cell Growth in vitro Sing G.K.;Keller J.R.;Ellingswqorth L.R.;Rusceni F.W.
  11. Cell v.61 TGF- β1 inhibition of c-myc transcription and growth in keratinocytes is abrogated by viral transforming protein with pRB binding domains Pietenpol J.A.;Stein R.W.;Moran E.;Yaciuk P.;Schlegel R.;Lyons R.M.;pittelkow M.R.;Munger K.;Howley P.M.;Moses H.L.
  12. Cell v.62 Growth Inhibitor from BSC-1 Cells Closely Related to Type beta Transforming Growth Factor Laiho M.;DeCaprio J.A.;Ludlow J.W.;Livingston D.M.;Massague J.
  13. Natl. Cancer Inst v.81 Inhibition and Promotion of Diffe-rentiated-Like Phenotype of a Human Lung Carcinoma in Athymic Mice by Matural and Recombinant Forms of TGF - β Twardzik D.R.
  14. Hepatology v.23 TGF - β1-induced Cell Death in Preneoplastic Foci of Rat Liver and Sensitization by the Antiestrogen Tamoxifen Mullauer L.;Brasl-Kraupp B.;Bursch W.;Schulte-Hermann R.
  15. Cancer v.77 no.8 suppl TGF- β1 Induces Apoptosis in Gastric Cancer Cells through a P53-independent Pathway Yamamoto M.;Maehara Y.;Sakaguchi Y.;Kusumoto T.;Ichiyoshi Y;Sugimachi K.
  16. Br-J-Concer v.72 Relationship between Tamoxifen-induced TGF β1 Expression Perry R.R.;Kang Y.;Greaves B.R.
  17. Blood v.80 Hematopoietic Cytokines Inhitit Apoptosis by TGF β1 and Cancer Ch-emotherapy Compounds in Myeloid Leukemic Cells Lotem J.;Sachs L.
  18. J-Interferon-Cytokine-Res v.15 Inhitition of Human Immunodeficiency Virus Type 1Multiplication by TGF β1 and AZT in HIV-1-infected Myeloid Cells Mckiel V.;Gu Z.;Wainberg M.A.;Hiscott J.
  19. Growth Factors v.8 Transforming Growth Factor β: Latent Forms, Binding Proteins and Receptors Miyazono K.;Ichijo H.;Hedin C.
  20. Biochem. Biophys. Res. Commun v.133 Conversion of High Molecular Weight Latent beta-Transforming Growth Factor from Chicken Embryo Fibroblasts into a Low Molecular Weight Active β-Transforming Growth Factor under Acidic conditions Lawrence D.A.;Picher R.;Jullien P.
  21. Biochm. Biophy. Res. Comm v.157 A New Method for High Yield urification of Type beta Transforming Growth Factor from Human Platelets Van den E.R.A.J.M.;Koornneef I.;Van zoelen E.J.J.
  22. J. Biol. Chem. v.258 Transforming Growth Factor-β in Human Platelets: Identification of a Major Storage Site, Purification and Characterization Assoian R.K.;Komoriya A.;Meyers C.A.;Miller D.M.;Sporn M.B.
  23. Cell v.48 The Transforming Growth Factor System, a Complex Pattern of Cross-Reactive Ligands and Receptors Cheiifetz S.;Weatherbee J.A.;Tsang M.L.S.;Anderson J.K.;Mole J.E.;Mole J.E.;Lucas R.;Massague J.
  24. Biochm. Biophy. Res. Comm v.136 The Transforming Growth factor is Stored in Human Blood Platelets as a Latent High Molecular Weight Complex Percher R.;Jullien P.;Lawrence D.A.
  25. J. Biol. Chem. v.263 Latent Transforming Growth Factor β from Human Platelets Wakefield L.M.;Smith D.M.;Flanders K.C.;Sporn M.B.
  26. Biochem v.22 Purification and Properties of a Type beta Transforming Growth Factor from Bovine Kidney Roberts A.B.;Anzano M.A.;Meyers C.A.;Wideman J.;Blacher R.;Pan Y.C.E.;Stein S.;Lehrman R.;Smith J.M.;Lamb L.C.;Sporn M.B.
  27. Protein Expression and Purification v.4 Recombinant Human Transforming Growth Factor β1: Expression by Chinese Hamster Ovary Cells, Isolation, and Characterization Bourdrel L.