The Car9 affinity tag is a dodecameric silica-binding peptide that can be fused to the N- and C-termini of proteins of interest to enable their rapid and inexpensive purification on underivatized silica in a process that typically relies on l-lysine as an eluent. Here, we show that silica paper spin columns and borosilicate multi-well plates used for plasmid DNA purification are suitable for recovering Car9-tagged proteins with high purity in a workflow compatible with high-throughput experiments. Spin columns typically yield 100 μg of biologically active material that can be recovered in minutes with low concentrations of lysine. Because of their short bed length, spin columns also offer unique advantages, as evidenced by the selective recovery of functional Car9-tagged tobacco etch virus (TEV) protease from a fused and auto-cleaved maltose binding protein (MBP) folding partner that nonspecifically binds to silica in the presence of NaCl. These additional purification modalities should increase the versatility and appeal of the Car9 tag for affinity protein purification.

中文翻译：

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在硅胶衍生的旋转柱和96孔板上亲和纯化Car9标签的蛋白。

Car9亲和标签是一种十二聚体二氧化硅结合肽，可以与目标蛋白质的N和C末端融合，从而能够在通常依赖于l-赖氨酸作为洗脱液的过程中在衍生化的二氧化硅上进行快速廉价的纯化。在这里，我们显示了用于质粒DNA纯化的硅胶纸旋转柱和硼硅酸盐多孔板适用于在与高通量实验兼容的工作流程中回收具有高纯度的Car9标签蛋白质。离心柱通常会产生100μg的生物活性物质，可在数分钟内用低浓度的赖氨酸进行回收。由于床身长度短，旋转柱也具有独特的优势，如通过从融合的和自动切割的麦芽糖结合蛋白（MBP）折叠伴侣中选择性回收功能性Car9标记的烟草蚀刻病毒（TEV）蛋白酶所证明的那样，该折叠伴侣在NaCl存在下非特异性地与二氧化硅结合。这些额外的纯化方式应增加Car9标签对亲和蛋白纯化的多功能性和吸引力。