The College of Life Sciences, Nankai University, Tianjin 300071, China.
Biosci Trends. 2013 Apr;7(2):93-100.
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.
基质细胞衍生因子 Follistatin 样 1（FSTL1）已被证明通过作为拮抗剂负调控骨形态发生蛋白（BMP）/Smad1/5/8 信号通路，并与包括器官发生、免疫和心血管疾病在内的生理和病理事件有关。因此，它是潜在治疗干预研究的有吸引力的靶点。在这项研究中，我们在 Drosophila S2 细胞中建立了一个高水平的表达系统，该系统每升培养基可产生约 12.5 毫克重组鼠 Follistatin 样 1 蛋白（rFSTL1）。然后，使用 Ni-NTA 琼脂糖亲和层析和 HiLoad 16/60 Superdex 200 凝胶过滤进一步将重组蛋白纯化至大于 95%的纯度。通过其在培养的水貂肺上皮细胞中负调控 BMP/Smad1/5/8 信号通路的能力来评估 rFSTL1 的生物学活性。此外，我们使用坐滴蒸汽扩散法对含有 follistatin 样结构域的 rFSTL1 的截断形式进行了结晶。总之，我们已经产生并纯化了具有生物学活性的重组 FSTL1 蛋白，这对于进一步的蛋白质结构和药物发现研究将非常重要。
