Department of Chemical Engineering, University of Washington, Seattle, WA, 98195, USA; Department of Material Sciences and Engineering, University of Washington, Seattle, WA, 98195, USA.
Department of Chemical Engineering, University of Washington, Seattle, WA, 98195, USA.
Protein Expr Purif. 2020 Jun;170:105608. doi: 10.1016/j.pep.2020.105608. Epub 2020 Feb 14.
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.
Car9 亲和标签是一种十二聚体硅结合肽，可以融合到感兴趣的蛋白质的 N 和 C 末端，以在未衍生的硅胶上快速且廉价地纯化它们，该过程通常依赖 l-赖氨酸作为洗脱剂。在这里，我们表明，用于质粒 DNA 纯化的硅胶纸螺旋柱和硼硅酸盐多孔板适用于以与高通量实验兼容的工作流程从 Car9 标记的蛋白质中回收高纯度的蛋白质。螺旋柱通常可产生 100μg 的生物活性物质，可在低浓度赖氨酸存在的情况下在数分钟内回收。由于其短床长度，螺旋柱还具有独特的优势，这一点可以从功能 Car9 标记的烟草蚀纹病毒（TEV）蛋白酶的选择性回收中得到证明，该蛋白酶来自融合和自动切割的麦芽糖结合蛋白（MBP）折叠伴侣，在 NaCl 存在下，该伴侣会非特异性地与硅胶结合。这些额外的纯化方式应该会增加 Car9 标签用于亲和蛋白纯化的多功能性和吸引力。
