Sharma Swati, Guptasarma Purnananda
Protein Science and Engineering Division, Institute of Microbial Technology (IMTECH), Chandigarh, India.
FEBS Lett. 2008 Jun 25;582(15):2203-11. doi: 10.1016/j.febslet.2008.05.008. Epub 2008 May 20.
We describe the behavior of a polypeptide consisting of the genetic fusion of a structurally stable single-domain protein, EGFP (an analog of the green fluorescent protein) with an amyloidogenic sequence, retroCspA (known to readily form amyloid fibrils). Refolding of the fusion protein through single-step removal of denaturant and salt results in precipitation into amyloid aggregates displaying fibrillar morphology, thioflavin T binding as well as green fluorescence. Refolding through step-wise reduction of denaturant concentration in the presence of salt yields a soluble aggregate containing a folded, thermally-stable, non-fluorescent EGFP domain. Together, these results indicate that retroCspA forces the fusion protein to aggregate; however, the EGFP domain remains folded in a native-like structural format in both soluble aggregates and precipitates.
我们描述了一种多肽的行为，该多肽由结构稳定的单域蛋白EGFP（绿色荧光蛋白的类似物）与淀粉样生成序列retroCspA（已知易形成淀粉样纤维）的基因融合而成。通过一步去除变性剂和盐使融合蛋白复性，导致其沉淀形成呈现纤维形态、具有硫黄素T结合以及绿色荧光的淀粉样聚集体。在有盐存在的情况下通过逐步降低变性剂浓度进行复性，产生一种可溶性聚集体，其中包含折叠的、热稳定的、无荧光的EGFP结构域。总之，这些结果表明retroCspA促使融合蛋白聚集；然而，EGFP结构域在可溶性聚集体和沉淀物中均以类似天然的结构形式折叠。
