Scalable, Non-denaturing Purification of Phosphoproteins Using Ga3+-IMAC: N2A and M1M2 Titin Components as Study case
Scalable, Non-denaturing Purification of Phosphoproteins Using Ga3+-IMAC: N2A and M1M2 Titin Components as Study case
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DOI：
10.1007/s10930-019-09815-w
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发表时间：
2019-04-01
期刊：
影响因子：
3
通讯作者：
Mayans, Olga
中科院分区：
文献类型：
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作者：
Adams, Michael;Fleming, Jennifer R.;Mayans, Olga
为了进行生化或生物物理分析，以折叠状态和足够数量的磷酸化蛋白质的提纯仍然是一项具有挑战性的任务。在这里，我们开发了一种新的实现方法的镓固定金属层析(GA3+-IMAC)，以允许选择性浓缩磷蛋白在毫克级和自然条件下使用自动化FPLC仪器。我们将这种方法应用于肌肉蛋白Titin被cAMP依赖的蛋白激酶(PKA)体外单磷酸化后的UN2A和M1M2组分的纯化。我们发现UN2A在其残基S9578的C末端被PKA磷酸化，而M1M2在其T32607位的结构域间接头序列中被磷酸化。我们证明了GA3+-IMAC方法是一种高效、经济的方法，适合在重组蛋白的自动化纯化管道中实施。该方法既可用于选择性浓缩，也可用于从生化样品中去除磷蛋白。
The purification of phosphorylated proteins in a folded state and in large enough quantity for biochemical or biophysical analysis remains a challenging task. Here, we develop a new implementation of the method of gallium immobilized metal chromatography (Ga3+-IMAC) as to permit the selective enrichment of phosphoproteins in the milligram scale and under native conditions using automated FPLC instrumentation. We apply this method to the purification of the UN2A and M1M2 components of the muscle protein titin upon being monophosphorylated in vitro by cAMP-dependent protein kinase (PKA). We found that UN2A is phosphorylated by PKA at its C-terminus in residue S9578 and M1M2 is phosphorylated in its interdomain linker sequence at position T32607. We demonstrate that the Ga3+-IMAC method is efficient, economical and suitable for implementation in automated purification pipelines for recombinant proteins. The procedure can be applied both to the selective enrichment and to the removal of phosphoproteins from biochemical samples.