毕赤酵母
重组DNA
转化生长因子β
大肠杆菌
转化生长因子
生物
转化生长因子β信号通路
分子生物学
细胞外
化学
生物化学
细胞生物学
基因
作者
Harrie L. Glansbeek,H.M. van Beuningen,E.L. Vitters,P.M. van der Kraan,Wim B. van den Berg
标识
DOI:10.1006/prep.1997.0819
摘要
Transforming growth factor-beta (TGF-beta) is a potent regulator of cell metabolism, proliferation, and differentiation. To study the role of endogenous TGF-beta in processes such as tissue repair and inflammation, potent and specific inhibitors are required. Because the type II TGF-beta receptor (TGF beta RII) has a high affinity for TGF-beta, the extracellular domain of TGF beta RII (TGF-beta sRII) was expressed in Pichia pastoris and Escherichia coli. Expression of the soluble TGF beta sRII using P. pastoris resulted in a soluble, heterogeneously glycosylated protein which was secreted into the medium. Although expression of TGF beta sRII in E. coli resulted in the formation of insoluble inclusion bodies, solubilization and refolding resulted in a biologically active protein. Because in both systems a C-terminal 6x His coding sequence was inserted behind the coding sequence for the extracellular domain of TGF beta RII the recombinant proteins could be purified by a powerful, single-step procedure using a Ni-NTA agarose. The purified proteins appeared to be potent inhibitors of TGF-beta 1 and TGF-beta 3. In contrast, TGF beta sRII was less effective in neutralization of TGF-beta 2. In conclusion, biologically active TGF beta sRII can be produced using P. pastoris and E. coli expression systems. The ease of these expression systems, the powerful single step purification and low costs makes it possible to produce TGF beta s RII in large amounts to further elucidate the role of TGF-beta 1 and TGF-beta 3 in physiological processes like tissue repair and inflammation.
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