Panchapakesan Shanker Shyam Sundhar, Jeng Sunny C Y, Unrau Peter J
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA.
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada.
Bio Protoc. 2018 Apr 5;8(7):e2799. doi: 10.21769/BioProtoc.2799.
A native purification strategy using RNA Mango for RNA based purification of RNA-protein complexes is described. The RNA Mango aptamer is first genetically engineered into the RNA of interest. RNA Mango containing complexes obtained from cleared cellular native extracts are then immobilized onto TO1-Desthiobiotin saturated streptavidin agarose beads. The beads are washed to remove non-specific complexes and then the RNA Mango containing complexes are eluted by the addition of free biotin to the beads. Since the eluted complexes are native and fluorescent, a second purification step such as size exclusion chromatography can easily be added and the purified complexes tracked by monitoring fluorescence. The high purity native complexes resulting from this two-step purification strategy can be then used for further biochemical characterization.
本文描述了一种使用RNA Mango进行基于RNA的RNA-蛋白质复合物纯化的天然纯化策略。首先将RNA Mango适体基因工程改造到目标RNA中。然后将从澄清的细胞天然提取物中获得的含RNA Mango的复合物固定到TO1-脱硫生物素饱和的链霉亲和素琼脂糖珠上。洗涤珠子以去除非特异性复合物，然后通过向珠子中添加游离生物素洗脱含RNA Mango的复合物。由于洗脱的复合物是天然且有荧光性的，因此可以轻松添加第二步纯化步骤，如尺寸排阻色谱法，并通过监测荧光来追踪纯化的复合物。这种两步纯化策略产生的高纯度天然复合物可用于进一步的生化表征。
