de Marco Ario
University of Nova Gorica (UNG), Rožna Dolina (Nova Gorica), Slovenia.
Methods Mol Biol. 2011;752:1-15. doi: 10.1007/978-1-60327-223-0_1.
The yields of soluble recombinant protein expressed in bacteria can be significantly enhanced by optimally exploiting the cell-folding machinery. The proposed protocol describes the strategies that can be used to reach a suitable ratio between heat-shock proteins and target proteins. Specifically, molecular recombinant chaperones can be overexpressed or cell-native chaperones are stimulated by inducing chemical and physical stress. Furthermore, the protein synthesis block can make available the cell-folding machinery for in vivo, disaggregating and refolding the already produced misfolded recombinant target protein. A rapid fluorimetric analytical method allows the evaluation of the protein monodispersity at any single purification step and enables comparison of different growth combinations that are useful to test for screening the optimal conditions for each recombinant protein.
通过最佳利用细胞折叠机制，可显著提高细菌中表达的可溶性重组蛋白产量。所提出的方案描述了可用于实现热休克蛋白与靶蛋白之间合适比例的策略。具体而言，可过表达分子重组伴侣蛋白，或通过诱导化学和物理应激来刺激细胞内源性伴侣蛋白。此外，蛋白质合成阻断可使细胞折叠机制用于体内分解和重折叠已产生的错误折叠重组靶蛋白。一种快速荧光分析方法可在任何单个纯化步骤评估蛋白质的单分散性，并能够比较不同的生长组合，这有助于测试筛选每种重组蛋白的最佳条件。
