A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning.
A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning.
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DOI：
10.1038/nprot.2008.250
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发表时间：
2009
期刊：
影响因子：
14.8
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中科院分区：
文献类型：
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蜘蛛丝的极端强度和弹性源于其重复蛋白质的模块化特性。为了开发这种材料和模拟蜘蛛丝，需要综合的策略来生产和纺重组纤维蛋白。本协议描述了丝基因的设计和克隆，蛋白在细菌中的表达，重组蛋白的纯化和纤维的形成。通过改进的基因构建和克隆方案，该技术适用于任何重复纤维蛋白的生产，并确保了天然重复序列，类似物或嵌合版本的精确复制。将蛋白质以25-30% （wt/vol）溶解于1,1,1,3,3,3-六氟-2-丙醇（HFIP）中，以挤压成纤维。该方案通常用于从不同蜘蛛物种的几种重组丝样蛋白中纺成单个微米大小的纤维，是一个强大的工具，可以生成具有相应纤维的蛋白质文库，用于蛋白质基生物材料的结构-功能关系研究。该方案可在40 d内完成。
The extreme strength and elasticity of spider silks originate from the modular nature of their repetitive proteins. To exploit such materials and mimic spider silks, comprehensive strategies to produce and spin recombinant fibrous proteins are necessary. This protocol describes silk gene design and cloning, protein expression in bacteria, recombinant protein purification and fiber formation. With an improved gene construction and cloning scheme, this technique is adaptable for the production of any repetitive fibrous proteins, and ensures the exact reproduction of native repeat sequences, analogs or chimeric versions. The proteins are solubilized in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at 25–30% (wt/vol) for extrusion into fibers. This protocol, routinely used to spin single micrometer-size fibers from several recombinant silk-like proteins from different spider species, is a powerful tool to generate protein libraries with corresponding fibers for structure–function relationship investigations in protein-based biomaterials. This protocol may be completed in 40 d.