Self-assembly of a designed protein polymer into β-sheet fibrils and responsive gels
Self-assembly of a designed protein polymer into β-sheet fibrils and responsive gels
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DOI：
10.1021/ma025952z
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发表时间：
2003-04-22
期刊：
影响因子：
5.5
通讯作者：
Muller, SJ
中科院分区：
文献类型：
--
作者：
Goeden-Wood, NL;Keasling, JD;Muller, SJ
构建了编码氨基酸重复序列的人工基因AEAEAKAKAEAEAKAK(9)，并在大肠杆菌中表达。提供17236个Da his标记的融合蛋白poly-EAK9。圆二色性和FTIR结果表明，聚eak9在反平行β -片超分子聚集体中采用延长的β -链构象。该结构表现出高度稳定和耐变性高达6 M尿素和在一定范围内的pH值和温度条件。刚果红染料结合实验支持淀粉样原纤维的存在，扫描电镜研究证实这些超分子结构实际上是光滑的，定义明确的原纤维，直径约10- 20nm。触发凝胶在生理条件下发生，表明聚eak9凝胶作为生物相容性和可生物降解材料的潜在用途。流变学和扫描电镜实验都证实了聚合物网的形成，其中聚合物网的大小与蛋白质浓度呈负相关。流变学测量表明，该材料比相同结构的低分子量合成肽具有更大的弹性。这些结果表明，可以设计出一种高分子量、复杂性最小的生物合成蛋白质聚合物，使其在溶液中自组装，形成β片原纤维和透明的自支撑凝胶。
An artificial gene encoding the amino acid repeat sequence (AEAEAKAKAEAEAKAK)(9) was constructed and expressed in E. coli. affording a 17 236 Da His-tagged fusion protein, poly-EAK9. Circular dichroism and FTIR results suggest that poly-EAK9 adopts an extended beta-strand conformation in an antiparallel beta-sheet supramolecular aggregate. The structure appears highly stable and resistant to denaturation up to 6 M urea and over a range of pH and temperature conditions. Congo Red dye-binding assays support the presence of amyloid-like fibrils, and scanning electron microscopy studies confirm that these supramolecular structures are in fact smooth, well-defined fibrils, approximately 10-20 nm in diameter. Triggered gelation occurs under physiological conditions, suggesting the potential use of poly-EAK9 gels as biocompatible and biodegradable materials. Rheological and SEM experiments are consistent with the formation of an entangled polymer network, in which the polymer mesh size appears to be inversely related to protein concentration. Rheological measurements indicate that the material is significantly more elastic than a lower molecular weight synthetic peptide of the same structure. These results demonstrate that a high molecular weight, biosynthetic protein polymer of minimal complexity can be designed to self-assemble in solution to form beta-sheet fibrils and transparent, self-supporting gels.