Expression, purification, refolding, and characterization of recombinant human soluble-Fas ligand from Escherichia coli

Kuang Hui Sun, Guang Huan Sun, Chang Youh Tsai, Hsiao Hsien Wang, Chang Chung-I, Guang Lin, Wen Wen Lin, Shye Jye Tang*

*此作品的通信作者

研究成果: Article同行評審

3 引文 斯高帕斯(Scopus)

摘要

Fas ligand (FasL) is a type II membrane protein and its biologically active soluble form (sFasL) is made by matrix metalloproteinase cleavage. sFasL may induce apoptosis in the Fas-bearing cells and this special biological activity may be applied to cancer therapy. However, it is difficult to obtain recombinant sFasL because of the trimeric form of the protein. In this study, sFasL DNA was fused to the gene of thioredoxin at the amino-terminal and His-tag at the carboxyl terminal before overexpression in Escherichia coli. To restrict the degree of freedom in the subsequent refolding process, recombinant sFasL was dissolved in 6 M urea and then immobilized with nickel resin under the denaturing condition. The immobilized recombinant sFasL was incubated with excess denatured sFasL to perform refolding process by dilution under stepwise conditions. After the refolding procedures, stable trimers of sFasL oligomers formed and were identified by gel filtration, co-immunoprecipitation and Western blotting. The sFasL showed biological activities in inducing apoptosis in Fas-expressing T cell and glial cell lines. These findings indicate that preparation of trimer forms of sFasL from E. coli is feasible and this strategy may be useful for large-scale production of sFasL.

原文English
頁(從 - 到)527-534
頁數8
期刊Enzyme and Microbial Technology
36
發行號4
DOIs
出版狀態Published - 2 3月 2005

指紋

深入研究「Expression, purification, refolding, and characterization of recombinant human soluble-Fas ligand from Escherichia coli」主題。共同形成了獨特的指紋。

引用此