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首页> 外文期刊>The Analyst: The Analytical Journal of the Royal Society of Chemistry: A Monthly International Publication Dealing with All Branches of Analytical Chemistry >Kinase-loaded magnetic beads for sequential in vitro phosphorylation of peptides and proteins
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Kinase-loaded magnetic beads for sequential in vitro phosphorylation of peptides and proteins

机译：激酶加载的磁珠，用于连续体外磷酸化肽和蛋白质

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摘要

Post-translational modifications, including phosphorylation, greatly impact the physiological function of proteins, especially those that are natively unfolded and implicated in many neurodegenerative diseases. However, structural and functional studies of such proteins require fully defined phosphorylation, including those that are not physiological. Thus, the kinases ERK2 and GSK-3 beta were immobilized to various superparamagnetic beads with carboxylic, aldehyde, Ni2+, or Co3+ functional groups, with a view to efficiently phosphorylate peptides and proteins in vitro. Full phosphorylation of specific synthetic peptides confirmed that beads were successfully loaded with kinases. Remarkably, enzymes covalently immobilized on carboxylated SeraMag beads remained active upon reuse, with residual activity after 10 uses 99.5 +/- 0.34% for GSK-3 beta and 36.2 +/- 2.01% for ERK2. The beads were also used to sequentially phosphorylate recombinant tau, which in vivo is a biomarker of Alzheimer's disease. Thus, a system consisting of two fully active kinases immobilized to magnetic beads is demonstrated for the first time. In comparison to soluble enzymes, the beads are easier to handle, reusable, and thus low-cost. Importantly, these beads are also convenient to remove from reactions to minimize contamination of phosphorylated products or to exchange with other kinases.
机译：翻译后修饰，包括磷酸化，大大影响蛋白质的生理功能，尤其是那些本身展开并涉及许多神经变性疾病的生理功能。然而，这种蛋白质的结构和功能研究需要完全定义的磷酸化，包括那些不是生理的。因此，将激酶ERK2和GSK-3β固定在羧酸，醛，Ni2 +或CO 3 +官能团上的各种超顺磁珠固定，以有效地磷酸化肽和体外蛋白质。特异性合成肽的全磷酸化证实，用激酶成功地装载珠子。值得注意的是，在重用后，共价固定在羧化血清珠粒上的酶在再利用后，10次使用109.5 +/- 0.34％的残留活性，ERK2的36.2 +/- 2.01％。珠子还用于依次磷酸化重组Tau，其在体内是阿尔茨海默病的生物标志物。因此，首次对由固定在磁珠上的两个完全活性激酶组成的系统。与可溶性酶相比，珠子更容易处理，可重复使用，从而更低。重要的是，这些珠子也方便地除去反应，以最小化磷酸化产品的污染或与其他激酶交换。

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