Expression, characterization, and preliminary X-ray crystallographic analysis of recombinant murine Follistatin-like 1 expressed in Drosophila S2 cells.
Expression, characterization, and preliminary X-ray crystallographic analysis of recombinant murine Follistatin-like 1 expressed in Drosophila S2 cells.
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DOI：
10.5582/bst.2013.v7.2.93
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发表时间：
2013-04
影响因子：
5.5
通讯作者：
Lian Li;Xinxin Li;Xue Liu;Yingying Dong;Y. Geng;Xinqi Liu;W. Ning
中科院分区：
文献类型：
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作者：
Lian Li;Xinxin Li;Xue Liu;Yingying Dong;Y. Geng;Xinqi Liu;W. Ning
基质细胞蛋白卵泡抑素样蛋白1(Follistatin-like 1，FSTL1)作为一种拮抗剂对骨形态发生蛋白(BMP)/Smad1/5/8信号转导起负性调节作用，参与器官发生、免疫和心血管疾病等生理和病理过程。因此，它是潜在的治疗干预研究的一个有吸引力的靶点。在本研究中，我们建立了一个在果蝇S2细胞中高效表达的系统，每升培养基能产生约12.5 mg重组小鼠卵泡抑素样蛋白(RFSTL1)。重组蛋白经Ni-NTA琼脂糖亲和层析和HiLoad 16/60 Superdex 200凝胶过滤纯化，纯度达95%以上。RFSTL1对培养的水貂肺上皮细胞BMP/Smad1/5/8信号的负调控作用评价其生物学活性。此外，我们还利用静置滴气扩散法合成了含有卵泡抑素样结构域的截短形式的rFSTL1。综上所述，我们获得并纯化了具有生物活性的重组FSTL1蛋白，这将为进一步的蛋白质结构和药物发现研究奠定基础。
The matricellular protein Follistatin-like 1 (FSTL1) has been shown to negatively regulate bone morphogenetic protein (BMP)/Smad1/5/8 signaling by functioning as an antagonist and has been implicated in physiological and pathological events including organogenesis, immunity and cardiovascular disease. It is therefore an attractive target for potential therapeutic intervention studies. In this study, we established a high-level expression system in Drosophila S2 cells which could produce about 12.5 mg of recombinant murine Follistatin-like 1 protein (rFSTL1) per liter of culture medium. The recombinant protein was then purified to greater than 95% purity using Ni-NTA agarose affinity chromatography followed by HiLoad 16/60 Superdex 200 gel filtration. The biological activity of rFSTL1 was evaluated by its ability to negatively regulate BMP/Smad1/5/8 signaling in cultured mink lung epithelial cells. Furthermore, we crystallized a truncated form of rFSTL1 containing the follistatin-like domain using the sitting drop vapor diffusion method. In conclusion, we have generated and purified biologically active recombinant FSTL1 protein, which will be important for further protein structure and drug discovery studies.