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北京大學(xué)使用我司磁珠泛素和泛素化蛋白納米受體發(fā)表文章

Construction of nano receptors for ubiquitin and ubiquitinated proteins based on the region-specific interactions between ubiquitin and polydopamine?

基于泛素和聚多巴胺區(qū)域特異性相互作用構(gòu)建泛素和泛素化蛋白納米受體
Zezhou Li,?a   Xinyi Li,?a   Wei Xian,?b   Huaisyuan Xie,a   Ying Sun,a   Yuxuan Zhang,a   Jiayu Wang,a   Hongwei Li,a   Changwen Jin,a   Xiaoyun Liu,*b   Zhiwei Zhu 

Abstract
Ubiquitination is a prevalent post-translational modification that controls a multitude of important biological processes. Due to the low abundance of ubiquitinated proteins, highly efficient separation and enrichment approaches are required for ubiquitinome analysis. In this work, we disclose the region-specific interactions between the hydrophobic patch of ubiquitin and polydopamine. Taking advantage of this inherent binding property, we have constructed surface-imprinted magnetic nanoparticles (NPs) for ubiquitin by sequential dopamine polymerization and surface PEGylation. The obtained molecularly imprinted polymer (MIP) NPs showed a binding constant of 2.6 × 106 L mol?1 for the template ubiquitin. The bound ubiquitin could be quantitatively released by heating to 70 °C at pH 2.0 or 90 °C at neutral (pH 7.0) conditions. The MIP NPs exhibited nano receptor-like property which not only effectively blocked the formation of branched ubiquitin chains but also selectively separated ubiquitin from the bacterial cell lysates. By incubating the MIP NPs with the lysates of 293T cells, totally 529 ubiquitinated proteins were captured, among which 287 proteins were not identified by the anti-ubiquitin monoclonal antibodies (mAbs). With the distinct merits of low cost and high stability, the as-prepared MIP NPs may be utilized either separately or as an important complement to the mAbs for the purification and enrichment of ubiquitin and ubiquitinated proteins from complex biological samples. Furthermore, due to the flexibility in modification of the binding sites during or after the imprinting reactions, the results of this work also paved the way for generation of artificial receptors for branched ubiquitin chains and polyubiquitinated proteins with higher avidity and specificity.

泛素化是一種普遍的翻譯后修飾，它控制著許多重要的生物過(guò)程。由于泛素化蛋白質(zhì)的豐度低，泛素組分析需要高效的分離和富集方法。在這項(xiàng)工作中，我們揭示了泛素疏水貼片和聚多巴胺之間的區(qū)域特異性相互作用。利用這種固有的結(jié)合特性，我們通過(guò)順序多巴胺聚合和表面聚乙二醇化構(gòu)建了泛素的表面印跡磁性納米粒子 (NPs)。獲得的分子印跡聚合物 (MIP) NPs 對(duì)模板泛素的結(jié)合常數(shù)為 2.6 × 106 L mol-1。結(jié)合的泛素可以通過(guò)在 pH 2.0 下加熱至 70 °C 或在中性 (pH 7.0) 條件下加熱至 90 °C 來(lái)定量釋放。 MIP NPs表現(xiàn)出類似納米受體的特性，不僅有效地阻止了分支泛素鏈的形成，而且還選擇性地將泛素從細(xì)菌細(xì)胞裂解物中分離出來(lái)。通過(guò)將 MIP NPs 與 293T 細(xì)胞的裂解物一起孵育，共捕獲了 529 個(gè)泛素化蛋白質(zhì)，其中 287 個(gè)蛋白質(zhì)未被抗泛素單克隆抗體 (mAb) 鑒定。由于具有低成本和高穩(wěn)定性的獨(dú)特優(yōu)點(diǎn)，所制備的 MIP NP 可以單獨(dú)使用，也可以作為 mAb 的重要補(bǔ)充，用于從復(fù)雜的生物樣品中純化和富集泛素和泛素化蛋白質(zhì)。此外，由于在印跡反應(yīng)期間或之后修飾結(jié)合位點(diǎn)的靈活性，這項(xiàng)工作的結(jié)果也為產(chǎn)生具有更高親和力和特異性的分支泛素鏈和多泛素化蛋白的人工受體鋪平了道路。