Bioorthogonal site-selective protein immobilisation and labelling

最近，我们发现一种酶（PPTase）可以用来将用小标签修饰的蛋白质附着在一系列不同的材料上，这些材料含有天然产生的分子辅酶a。这种方法温和而高效，可以使蛋白质以均匀的方向附着在表面上，从而保持高活性。考虑到附着在蛋白质上的短标签在结构上是独特的，可以直接从细胞提取物中固定蛋白质，而不需要耗时地分离纯形式的感兴趣的蛋白质。然而，由于细胞自身的辅酶A的存在，这种从细胞提取物中固定的效率降低了，辅酶A可以竞争附着在标记的蛋白质上。在这个项目中，我们将设计更有效的PPTases，不再识别天然辅酶A，而是针对人造类似物。我们设想，新的PPTase酶将是将大量标记蛋白直接从复杂的细胞混合物快速附着到芯片（玻片）上的理想选择。使用这种蛋白质芯片（或阵列），可以在单个芯片上同时表征大量蛋白质，只需少量的材料。事实上，蛋白质芯片可以用来筛选与固定蛋白质结合的其他生物分子，这可以帮助解决与蛋白质功能有关的基本生物学问题。固定的蛋白质也可以用于药物筛选、诊断和作为检测设备的一部分。迄今为止，蛋白质阵列仅限于相对有限的蛋白质数量。然而，据估计，仅在人类身上，就可能有数十万种独特的蛋白质。未来蛋白质阵列的产生可能容纳更多的蛋白质，需要高效的蛋白质固定方法和芯片表面蛋白质特征的进一步小型化。鉴于此，我们将结合并利用我们新的蛋白质固定方法和最先进的纳米制造方法，这种方法允许在玻片上产生纳米化学“斑点”（直径小于十亿分之一米），我们将在其上附着蛋白质。然后，我们将通过研究蛋白质与其他蛋白质、DNA和药物分子的相互作用，来证明附着在纳米级特征上的蛋白质的活性。除了蛋白质固定化，我们开发的新的更有效的PPTase酶也可以用于蛋白质的位点特异性标记。荧光标记、药物和其他分子附着在蛋白质上对于研究蛋白质功能和开发基于蛋白质的药物（生物制药）也很重要。

Recently, we discovered that an enzyme (PPTase) can be used to attach proteins, which have been modified with a small tag, to a range of different materials that present the naturally produced molecule coenzyme A. This method is mild and efficient and enables the site-specific attachment of proteins in a uniform orientation on to a surface, thus maintaining high activity. Given that the short tag attached to the protein is unique in structure, it is possible to immobilise proteins directly from cell extracts without the need for time-consuming isolation of the protein of interest in pure form. However, the efficiency of this immobilisation from cellular extracts is reduced due to the presence of the cells' own coenzyme A, which can compete for attachment to the tagged protein. In this project we will engineer PPTases that are more efficient and no longer recognise the natural coenzyme A, but instead are specific for man-made analogues. We envisage that the new PPTase enzymes will be ideal for the rapid attachment of large numbers of tagged proteins on to chips (glass slides) directly from complex cellular mixtures. Using such protein chips (or arrays) it is possible to characterise large numbers of proteins simultaneously on a single chip, with minute amounts of material. Indeed protein chips can be used to screen for other biomolecules that bind to the immobilised proteins, which can help address fundamental biological questions relating to protein function. Immobilised proteins can also be used for drug screening, diagnostics and as a part of detection devices. To date, protein arrays have been limited to relatively modest numbers of proteins. However it is estimated that in humans alone, there could be hundreds of thousands of unique proteins. The generation of future protein arrays, which might accommodate larger numbers proteins, requires highly efficient protein immobilisation methodology and further miniaturisation of protein features on the surface of chips. In light of this we will combine and exploit our new protein immobilisation method with state-of-the-art nanofabrication methods which allow nanometre chemical 'spots' (less than a billionth of a meter in diameter) to be generated on glass slides, on to which we will attach proteins. We will then demonstrate the activity of the proteins attached to the nanoscale features, by studying their interactions with other proteins, DNA and drug molecules. In addition to protein immobilisation the new more efficient PPTase enzymes we develop can also be used in the site-specific labelling of proteins. The attachment of fluorescent labels, drugs, and other molecules to proteins is also important for studying protein function and in the development of protein based drugs (biopharmaceuticals).