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首页> 美国卫生研究院文献>Protein Science : A Publication of the Protein Society >Chemical denaturation and elevated folding temperatures are required for wild-type activity and stability of recombinant Methanococcus jannaschii 20S proteasome
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Chemical denaturation and elevated folding temperatures are required for wild-type activity and stability of recombinant Methanococcus jannaschii 20S proteasome

机译：变性甲烷球菌20S蛋白酶体的野生型活性和稳定性需要化学变性和升高的折叠温度

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摘要

The 20S proteasome from the extreme thermophile Methanococcus jannaschii (Mj) was purified and sequenced to facilitate production of the recombinant proteasome in E. coli. The recombinant proteasome remained in solution at a purity level of 80–85% (according to SDS PAGE) following incubation of cell lysates at 70°C. Temperature–activity profiles indicated that the temperature optima of the wild-type and recombinant enzymes differed substantially, with optimal activities occurring at 119°C and 95°C, respectively. To ameliorate this discrepancy, two recombinant enzyme preparations were produced, each of which included denaturation of the proteasome by 4 M urea followed by high-temperature (85°C) dialysis. The wild-type temperature optimum was restored, but only if proteasome subunits were denatured and refolded prior to assembly (a preparation designated as α & β). In contrast, when proteasome assembly preceded denaturation (designated α + β) the optimum temperature was raised to a lesser degree. Moreover, the α & β and α + β preparations had apparent thermal half-lives at 114°C of 54.2 and 26.2 min, respectively, and the thermostability of the less stable enzyme was more sensitive to a reduction in pH. Attainment of wild-type activity and stability thus required the proper folding of both the α- and β-subunits prior to proteasome assembly. Consistent with this behavior, dual-scanning calorimetry (DSC) measurements revealed differences in the reassembly efficiency of the two proteasome preparations. The ability to produce structural conformers with dramatically different thermal optima and thermostabilities may facilitate the determination of molecular forces and structural motifs responsible for enzyme thermostablity and high-temperature activity.
机译：来自极端嗜热甲烷球菌（Mj）的20S蛋白酶体经过纯化和测序，以促进在大肠杆菌中生产重组蛋白酶体。在70°C下孵育细胞裂解液后，重组蛋白酶体以80-85％的纯度（根据SDS PAGE）保留在溶液中。温度-活性曲线表明，野生型和重组酶的最适温度存在显着差异，最佳活性分别出现在119°C和95°C。为了改善这种差异，生产了两种重组酶制剂，每种制剂包括用4 M尿素使蛋白酶体变性，然后进行高温（85°C）透析。恢复了野生型最适温度，但前提是必须在组装前将蛋白酶体亚基变性并重新折叠（标为α和β的制剂）。相反，当蛋白酶体组装在变性之前（称为α+β）时，最佳温度升高的程度较小。此外，α和β制剂以及α+β制剂在114℃的表观热半衰期分别为54.2和26.2分钟，稳定性较差的酶的热稳定性对pH降低更敏感。因此，要获得野生型的活性和稳定性，就需要在蛋白酶体组装之前适当折叠α-亚基和β-亚基。与此行为一致，双扫描量热法（DSC）测量揭示了两种蛋白酶体制剂的重组效率差异。产生具有极大不同的热最佳和热稳定性的结构构象异构体的能力可以促进确定负责酶热稳定性和高温活性的分子力和结构基序。

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