Protocol for preparing proteins with improved solubility by co-expressing with molecular chaperones in Escherichia coli
Protocol for preparing proteins with improved solubility by co-expressing with molecular chaperones in Escherichia coli
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DOI：
10.1038/nprot.2007.400
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发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
de Marco, Ario
中科院分区：
文献类型：
--
作者：
de Marco, Ario
分子伴侣的八种组合（e.例如，在一个实施例中，DnaK/DnaJ/GrpE/ClpB）与目标重组蛋白共表达，以比较它们在提高其可溶性产量方面的有效性。该系统允许迄今为止提出的最完整和最合理的方法来使用分子伴侣活性来优化宿主细胞折叠机器。此外，一个两步的协议，其中蛋白质的合成和蛋白质的复性是解耦。分子伴侣和靶蛋白在第一生长期积累，然后靶蛋白聚集体在蛋白质合成受阻后在体内解聚。使特定蛋白质的可溶性产量最大化的最佳伴侣蛋白组合仍然不可预测。因此，小规模纯化选择步骤可用于在放大生产之前在表达组合中进行筛选。应用这种策略，我们可以增加70%的测试构建体的溶解度，其蛋白质产量比对照组高出42倍。整个过程需要2 d。
Eight combinations of molecular chaperones ( e. g., DnaK/DnaJ/GrpE/ClpB) are co-expressed with the target recombinant protein to compare their effectiveness in improving its soluble yield. This system allows the most complete and rational approach proposed so far to use the chaperone activity for optimizing the host cell folding machinery. Furthermore, a two-step protocol is presented, in which protein synthesis and protein refolding are uncoupled. Molecular chaperones and target protein accumulate in the first growth phase and target protein aggregates are then disaggregated in vivo after the block of protein synthesis. The optimal chaperone combination to maximize the soluble yield of a specific protein remains unpredictable. Therefore, a small-scale purification selection step is useful for screening among expression combinations before scaling-up production. Applying such a strategy, we could increase the solubility of 70% of the tested constructs with yields of up to 42-fold more protein than in controls. The procedure takes 2 d.