Immobilized proteins hold promise as the basic units that have enabled a broad range of analytical applications within chemical measurement science. As yet, the co-immobilization of diverse proteins at precise ratio and whether they give rise to improved analytical performance remain challengeable. Herein, we utilized a circularly permuted HaloTag (cpHaloTag) to achieve the co-immobilization of two proteins at precise ratio, which was applied in developing a chromatographic method with improved specificity for pursuing dual-target compounds. The methodology involved the fusion 3A and 2C at N- and C-terminuses of cpHaloTag, the immobilization of the fusion protein onto silica gel through bioorthogonal reaction, the morphological and functional characterization, the application in finding dual-target compounds. Expression of the fusion protein in system showed a yield of milligram level with the presence of 3A and 2C domains. Immobilization of the protein was achieved in 10 min with a reaction efficiency more than 88.5 %. Immobilized 3A-cpHalo-2C exhibited higher specificity and better retentions of canonical compounds of the two enzymes in comparison with the column containing immobilized 3A or 2C alone. In real sample application, screening analysis found that hyperoside, cymaroside, and baicalin were dual-target compounds in concert with 3A and 2C in extract. Taking 3A and 2C as probe, we proposed a simple method for direct co-immobilization of diverse proteins from cell lysates and demonstrated an affinity chromatographic-based dual-target compound screening platform. The implications of these methodology are possible to insight the de novo design of multi-target surface for fabricating new bioanalytical methods with improved performance.

中文翻译：

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以等摩尔比共固定两种不同蛋白质的自催化策略：以 3A 和 2C 为例，开发一种从复杂基质中寻找潜在双靶点化合物的色谱方法


固定化蛋白质有望作为基本单元，在化学测量科学中实现广泛的分析应用。迄今为止，以精确比例共固定不同蛋白质以及它们是否能提高分析性能仍然是一个挑战。在此，我们利用循环排列的HaloTag（cpHaloTag）实现了两种蛋白质以精确比例的共固定，用于开发一种具有更高特异性的色谱方法来寻找双靶点化合物。该方法涉及cpHaloTag N端和C端的3A和2C融合、通过生物正交反应将融合蛋白固定在硅胶上、形态和功能表征以及在寻找双靶点化合物中的应用。融合蛋白在系统中的表达显示出毫克级的产量，并且存在3A和2C结构域。 10分钟内实现蛋白质固定化，反应效率超过88.5%。与单独含有固定化 3A 或 2C 的柱相比，固定化 3A-cpHalo-2C 对两种酶的典型化合物表现出更高的特异性和更好的保留。在实际样品应用中，筛选分析发现金丝桃苷、西马苷、黄芩苷与提取物中的3A、2C配合，为双靶点化合物。以3A和2C为探针，我们提出了一种直接共固定细胞裂解液中多种蛋白质的简单方法，并展示了基于亲和色谱的双靶点化合物筛选平台。这些方法的含义可以洞察多目标表面的从头设计，以制造具有改进性能的新生物分析方法。