Tyler Robert C, Sreenath Hassan K, Singh Shanteri, Aceti David J, Bingman Craig A, Markley John L, Fox Brian G
Department of Biochemistry, Center for Eukaryotic Structural Genomics, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706-1549, USA.
Protein Expr Purif. 2005 Apr;40(2):268-78. doi: 10.1016/j.pep.2004.12.024.
Protocols have been developed and applied for the high-throughput production of [U-15N]- or [U-13C-, U-15N]-labeled proteins using the conditional methionine auxotroph Escherichia coli B834. The large-scale growth and expression uses a chemically defined auto-induction medium containing salts and trace metals, vitamins including vitamin B12, and glucose, glycerol, and lactose. The results from nine expression trials in 2-L of the auto-induction medium (500 mL in each of four polyethylene terephthalate beverage bottles) gave an average final optical density at 600 nm of approximately 5, an average wet cell mass yield of approximately 9.5 g L(-1), and an average yield of approximately 20 mg of labeled protein in the six instances in which proteolysis of the fusion protein was observed. Correlations between the cell mass recovered, the level of protein expression, and the relative amounts of glucose, glycerol, and lactose in the auto-induction medium were noted. Mass spectral analysis showed that the purified proteins contained both 15N and 13C at levels greater than 95%. 1H-15N heteronuclear single quantum correlation spectroscopy as well as 13C; 15N-edited spectroscopy showed that the purified [U-15N]- and [U-13C, U-15N]-labeled proteins were suitable for structure analysis.
已经开发并应用了一些方案，用于使用条件性甲硫氨酸营养缺陷型大肠杆菌B834高通量生产[U-15N]或[U-13C、U-15N]标记的蛋白质。大规模培养和表达使用一种化学成分明确的自诱导培养基，该培养基含有盐和痕量金属、包括维生素B12在内的维生素，以及葡萄糖、甘油和乳糖。在2升自诱导培养基（四个聚对苯二甲酸乙二醇酯饮料瓶中各500毫升）中进行的九次表达试验结果显示，600纳米处的平均最终光密度约为5，平均湿细胞质量产量约为9.5克/升，在观察到融合蛋白发生蛋白水解的六个实例中，标记蛋白的平均产量约为20毫克。注意到回收的细胞质量、蛋白质表达水平与自诱导培养基中葡萄糖、甘油和乳糖的相对含量之间的相关性。质谱分析表明，纯化后的蛋白质中15N和13C的含量均大于95%。1H-15N异核单量子相关光谱以及13C；15N编辑光谱表明，纯化后的[U-15N]和[U-13C、U-15N]标记蛋白质适用于结构分析。
