Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Pardubice 532 10, Czech Republic.
Analyst. 2018 Jan 15;143(2):466-474. doi: 10.1039/c7an01508a.
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-3β were immobilized to various superparamagnetic beads with carboxylic, aldehyde, Ni, or Co 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-3β 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.
翻译后修饰，包括磷酸化，对蛋白质的生理功能有很大影响，尤其是那些天然未折叠且与许多神经退行性疾病有关的蛋白质。然而，对此类蛋白质的结构和功能研究需要完全确定的磷酸化，包括那些非生理性的磷酸化。因此，将激酶ERK2和GSK - 3β固定在具有羧基、醛基、镍或钴官能团的各种超顺磁性珠上，以期在体外有效地使肽和蛋白质磷酸化。特定合成肽的完全磷酸化证实珠子已成功负载激酶。值得注意的是，共价固定在羧基化SeraMag珠上的酶在重复使用后仍保持活性，GSK - 3β在10次使用后的残余活性为99.5±0.34%，ERK2为36.2±2.01%。这些珠子还用于依次磷酸化重组tau蛋白，tau蛋白在体内是阿尔茨海默病的生物标志物。因此，首次展示了一种由固定在磁珠上的两种完全活性激酶组成的系统。与可溶性酶相比，这些珠子更易于操作、可重复使用，因此成本较低。重要的是，这些珠子也便于从反应中去除，以尽量减少磷酸化产物的污染或与其他激酶进行交换。
