Membrane proteins are targets of most available pharmaceuticals, but they are difficult to produce recombinantly, like many other aggregation-prone proteins. Spiders can produce silk proteins at huge concentrations by sequestering their aggregation-prone regions in micellar structures, where the very soluble N-terminal domain (NT) forms the shell. We hypothesize that fusion to NT could similarly solubilize non-spidroin proteins, and design a charge-reversed mutant (NT*) that is pH insensitive, stabilized and hypersoluble compared to wild-type NT. NT*-transmembrane protein fusions yield up to eight times more of soluble protein in Escherichia coli than fusions with several conventional tags. NT* enables transmembrane peptide purification to homogeneity without chromatography and manufacture of low-cost synthetic lung surfactant that works in an animal model of respiratory disease. NT* also allows efficient expression and purification of non-transmembrane proteins, which are otherwise refractory to recombinant production, and offers a new tool for reluctant proteins in general.
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机译:膜蛋白是大多数可用药物的靶标,但它们像许多其他易于聚集的蛋白一样,很难重组生产。蜘蛛可以通过隔离胶束结构中易于聚集的区域来制造高浓度的丝蛋白,其中非常易溶的N末端结构域(NT)构成了外壳。我们假设与NT的融合可以类似地溶解非spidroin蛋白,并设计了与野生型NT相比pH不敏感,稳定且超溶的电荷反转突变体(NT *)。 NT *-跨膜蛋白融合物在大肠杆菌中的可溶性蛋白产量是与几种常规标签融合物的八倍。 NT *无需色谱即可实现跨膜肽纯化的均质性,并可以生产在呼吸系统疾病动物模型中起作用的低成本合成肺表面活性剂。 NT *还可以有效表达和纯化非跨膜蛋白,否则重组生产将难以耐受,并提供了一种新的工具来检测总体上不需要的蛋白。
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