Protein expression in E. coli grown in shaker flasks is a routine and pivotal tool in many research laboratories. To maximize protein yields, cells are normally induced in the middle of the linear growth phase, typically at an OD600 of ≤ 1 for cells grown in Luria-Bertani (LB) medium at 37 °C. We recently showed that the E. coli linear growth phase can be extended to higher cell density when cells are cultured under less than optimal conditions such as in minimal medium and/or at lower temperatures. Maximizing the yield of protein per unit volume of culture is important for reducing the costs, especially when isotopically labeling is required. Here, we present a modified minimal medium and a simple protocol that can increase the protein yield up to fourfold in a pH-stabilized LB medium and up to sevenfold in a modified M9+ medium (M9++). When M9++ medium coupled with the high density (OD600 ~ 6) cell growth protocol are used to express uniformly 15N- or 15N/13C-labeled proteins, the amount of 15NH4Cl and 13C6-glucose for a given cell mass is reduced by 50% and ~ 65%, respectively, relative to the traditional low density (OD600 ~ 1) cell growth protocol with M9 medium; the inclusion of 0.1% LB in the minimal medium permits a reduction in the concentration of both the trace element solution and MgCl2, which can cause precipitation. Mass data indicate that inclusion of 0.1% LB does not significantly affect the isotope enrichment level.

中文翻译：

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用于在摇瓶中以高细胞密度生长的大肠杆菌中表达同位素标记蛋白的简单方案。

在摇瓶中培养的大肠杆菌中的蛋白质表达是许多研究实验室的常规且关键的工具。为了最大限度地提高蛋白质产量，通常在线性生长期的中期诱导细胞，对于在 37 °C 的 Luria-Bertani (LB) 培养基中生长的细胞，通常 OD600 ≤ 1。我们最近表明，当细胞在非最佳条件（例如基本培养基和/或较低温度）下培养时，大肠杆菌线性生长期可以延长至更高的细胞密度。最大化每单位体积培养物的蛋白质产量对于降低成本非常重要，特别是在需要同位素标记时。在这里，我们提出了一种改良的基本培养基和一个简单的方案，可以将 pH 稳定的 LB 培养基中的蛋白质产量提高至四倍，将改良的 M9+ 培养基 (M9++) 中的蛋白质产量提高至七倍。当 M9++ 培养基与高密度 (OD600 ~ 6) 细胞生长方案结合使用来均匀表达 15N 或 15N/13C 标记的蛋白质时，给定细胞团中 15NH4Cl 和 13C6-葡萄糖的量减少了 50%，并且相对于使用 M9 培养基的传统低密度 (OD600 ~ 1) 细胞生长方案分别约为 65%；在基本培养基中加入 0.1% LB 可以降低微量元素溶液和 MgCl2 的浓度，从而导致沉淀。质量数据表明，加入 0.1% LB 不会显着影响同位素富集水平。相对于使用M9培养基的传统低密度（OD600~1）细胞生长方案；在基本培养基中加入 0.1% LB 可以降低微量元素溶液和 MgCl2 的浓度，从而导致沉淀。质量数据表明，加入 0.1% LB 不会显着影响同位素富集水平。相对于使用M9培养基的传统低密度（OD600~1）细胞生长方案；在基本培养基中加入 0.1% LB 可以降低微量元素溶液和 MgCl2 的浓度，从而导致沉淀。质量数据表明，加入 0.1% LB 不会显着影响同位素富集水平。