Department of Biomedical Engineering, 4 Colby Street, Tufts University, Medford, Massachusetts 02155, USA.
Biomacromolecules. 2009 Dec 14;10(12):3227-34. doi: 10.1021/bm900735g.
Resilin is a polymeric rubber-like protein secreted by insects to specialized cuticle regions, in areas where high resilience and low stiffness are required. Resilin binds to the cuticle polysaccharide chitin via a chitin binding domain and is further polymerized through oxidation of the tyrosine residues resulting in the formation of dityrosine bridges and assembly of a high-performance protein--carbohydrate composite material. We describe the mechanical, structural and biochemical function of chitin binding recombinant Drosophila melanogaster resilin. Various resilin constructs were cloned including the full length gene enabling Ni-NTA purification, as well as heat and salt precipitation for rapid and efficient purification. The binding isotherms and constants (K(d), B(max)) of resilin to chitin via its chitin binding domain were determined and displayed high affinity to chitin, implying its important role in the assembly of the resilin-chitin composite. The structural and elastic properties were investigated using Fourier transform infrared spectroscopy, circular dichroism, and atomic force microscopy with peroxidase cross-linked solid resilin materials. Generally, little structural organization was found by these biophysical methods, suggesting structural order was not induced by the dityrosine cross-links. Further, the elastomeric properties found from the full length protein compared favorably with the shorter resilin generated previously from exon 1. The unusual elastomeric behavior of this protein suggests possible utility in biomaterials applications.
弹性蛋白是一种高分子橡胶状蛋白，由昆虫分泌到专门的表皮区域，在这些区域需要高弹性和低刚性。弹性蛋白通过一个几丁质结合结构域与几丁质多糖结合，并通过酪氨酸残基的氧化进一步聚合，形成二酪氨酸桥，组装成一种高性能的蛋白质-碳水化合物复合材料。我们描述了几丁质结合重组果蝇弹性蛋白的机械、结构和生化功能。克隆了各种弹性蛋白构建体，包括能够进行 Ni-NTA 纯化的全长基因，以及热和盐沉淀，以实现快速高效的纯化。通过其几丁质结合结构域测定了弹性蛋白与几丁质的结合等温线和常数（K(d)、B(max)），并显示出对几丁质的高亲和力，这表明它在弹性蛋白-几丁质复合材料的组装中具有重要作用。使用傅里叶变换红外光谱、圆二色性和原子力显微镜以及过氧化物酶交联的固态弹性蛋白材料研究了结构和弹性特性。通常，这些生物物理方法发现的结构组织很少，这表明结构顺序不是由二酪氨酸交联诱导的。此外，与之前从外显子 1 产生的较短弹性蛋白相比，全长蛋白发现的弹性特性较好。这种蛋白质不寻常的弹性行为表明它可能在生物材料应用中有一定的用途。
