Sreenath Hassan K, Bingman Craig A, Buchan Blake W, Seder Kory D, Burns Brendan T, Geetha Holalkere V, Jeon Won Bae, Vojtik Frank C, Aceti David J, Frederick Ronnie O, Phillips George N, 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):256-67. doi: 10.1016/j.pep.2004.12.022.
Protocols have been developed and applied in the high-throughput production of selenomethionine labeled fusion proteins using the conditional Met auxotroph Escherichia coli B834. The large-scale growth and expression uses a chemically defined auto-induction medium containing 125 mg L(-1) selenomethionine, salts and trace metals, other amino acids including 10 mg L(-1) of methionine, vitamins except vitamin B12, and glucose, glycerol, and alpha-lactose. A schematic for a shaker rack that can hold up to twenty-four 2-L polyethylene terephthalate beverage bottles in a standard laboratory refrigerated floor shaker is provided. The growth cycle from inoculation of the culture bottle through the growth, induction, and expression was timed to take approximately 24 h. Culture growth in the auto-induction medium gave an average final optical density at 600 nm of approximately 6 and an average wet cell mass yield of approximately 14 g from 2 L of culture in greater than 150 expression trials. A simple method for visual scoring of denaturing electrophoresis gels for total protein expression, solubility, and effectiveness of fusion protein proteolysis was developed and applied. For the favorably scored expression trials, the average yield of purified, selenomethionine-labeled target protein obtained after proteolysis of the fusion protein was approximately 30 mg. Analysis by mass spectrometry showed greater than 90% incorporation of selenomethionine over a approximately 8-fold range of selenomethionine concentrations in the growth medium, with higher growth rates observed at the lower selenomethionine concentrations. These protein preparations have been utilized to solve X-ray crystal structures by multiwavelength anomalous diffraction phasing.
已经开发出了相关方案，并应用于使用条件性甲硫氨酸营养缺陷型大肠杆菌B834高通量生产硒代甲硫氨酸标记的融合蛋白。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125 mg L(-1)硒代甲硫氨酸、盐和微量元素、包括10 mg L(-1)甲硫氨酸在内的其他氨基酸、除维生素B12外的维生素，以及葡萄糖、甘油和α-乳糖。提供了一种摇床架的示意图，该摇床架可在标准实验室冷藏落地式摇床中容纳多达24个2-L聚对苯二甲酸乙二醇酯饮料瓶。从接种培养瓶到生长、诱导和表达的生长周期经计时约为24小时。在超过150次表达试验中，自诱导培养基中的培养物生长在600 nm处的平均最终光密度约为6，2 L培养物的平均湿细胞质量产量约为14 g。开发并应用了一种简单的方法，用于对变性电泳凝胶上的总蛋白表达、溶解性和融合蛋白蛋白水解效果进行视觉评分。对于评分良好的表达试验，融合蛋白蛋白水解后获得的纯化的、硒代甲硫氨酸标记的靶蛋白的平均产量约为30 mg。质谱分析表明，在生长培养基中硒代甲硫氨酸浓度约8倍的范围内，硒代甲硫氨酸的掺入率大于90%，在较低硒代甲硫氨酸浓度下观察到更高的生长速率。这些蛋白质制剂已被用于通过多波长反常衍射相位法解析X射线晶体结构。
