The production of well-ordered crystals of membrane proteins for structural investigation by X-ray diffraction typically requires extensive crystallization trials and may involve the screening of multiple detergents, lipids and other additives. Purification of sufficient amounts of protein for such trials is hampered by the fact that even when over-expressed, membrane proteins represent only a small percentage of the total protein content of bacteria. Fermentation-scale cultures of cells are therefore usually required. To maximize the efficiency and reduce the cost of such cultures, in the UK Membrane Protein Structure Initiative we have systematically investigated the use of auto-induction as an alternative to induction of expression with isopropyl-beta-D-thiogalactoside. We report here the benefits of first optimizing expression on a multiwell plate scale by systematically varying the concentrations of glucose, glycerol, lactose and succinate present in the auto-induction medium. For subsequent scale-up, comparison of isopropyl-beta-D-thiogalactoside induction in shake-flasks with auto-induction in shake-flasks and in 1L fermenters without and with control of pH and aeration revealed that highest yields of target protein were obtained using the latter culture conditions. However, analysis of the time-course of expression highlighted the importance of choosing the correct time for harvest. The high yields of target protein that can be obtained in a single batch by auto-induction, performed on a 30 l scale in a fermenter, obviate batch-to-batch variations that can add an unwanted variable to crystallization screening experiments. The approach described should therefore be of great utility for membrane protein production for structural studies.

中文翻译：

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通过细菌自动诱导可靠地扩大膜蛋白的过表达范围：从微孔板到中试规模的发酵。

通过X射线衍射生产用于结构研究的膜蛋白井井有条的晶体通常需要进行广泛的结晶试验，并且可能涉及多种洗涤剂，脂质和其他添加剂的筛选。即使在过表达的情况下，膜蛋白也仅占细菌总蛋白含量的一小部分，这一事实妨碍了为此类试验纯化足够量的蛋白质。因此通常需要细胞的发酵规模培养。为了最大程度地提高此类培养物的效率并降低其成本，在英国膜蛋白结构计划中，我们系统地研究了使用自动诱导作为异丙基-β-D-硫代半乳糖苷诱导表达的替代方法。我们在这里报告了通过系统地改变自动诱导培养基中存在的葡萄糖，甘油，乳糖和琥珀酸盐的浓度，首先在多孔板规模上优化表达的好处。为了随后扩大规模，比较不加和控制pH值和曝气的摇瓶和1L发酵罐中的自动诱导的摇瓶中异丙基-β-D-硫代半乳糖苷诱导，发现使用以下方法可获得最高的目标蛋白产量后一种文化条件。但是，对表达时间过程的分析强调了选择正确的收获时间的重要性。可以在发酵罐中以30升规模通过自动诱导在单批中获得高产量的目标蛋白，消除了批次间的差异，该差异会为结晶筛选实验添加不必要的变量。因此，所描述的方法对于结构研究中的膜蛋白生产应具有很大的实用性。