Delta3D; Bench top assays for the rapid detection of protein 3D structural changes

药物曾经只是小分子，如阿司匹林和青霉素。它们分别含有21和42个原子，它们的结构由每个原子之间的刚性键决定。它们的纯度很容易测量，任何化学变化都可以通过既定的方法检测到，并且它们的生产是直接的。目前，新一代的生物制药正在出现，其使用大分子，例如在体内发现的那些，例如蛋白质，来选择性地治疗一系列疾病。最早的生物药物之一是胰岛素，从动物胰腺中提取，自20世纪20年代以来一直用于治疗因患者无法制造胰岛素而引起的糖尿病。没有一种小分子能像胰岛素那样起作用，现在药用胰岛素是由携带人类胰岛素基因的细菌制造的。胰岛素是一种小蛋白质，但即使如此，它也有791个原子，这些原子不仅通过共价键连接，而且还通过较弱的作用来稳定其复杂的3D结构。这说明了生物制药的主要特点：它们具有高度选择性的作用，具有巨大的医疗价值，但非常复杂，难以制造。目前有很多关于治疗以前无法治疗的疾病的药物的讨论，例如赫赛汀和转移性乳腺癌。赫赛汀是一种称为抗体的蛋白质，它可以杀死特定的癌细胞，就像我们自己的抗体保护我们免受入侵细胞一样。抗体目前占大多数新的生物药物，是超过20，000个原子的复合物。还有许多其他蛋白质用于靶向小分子无法实现的特定效果。这些包括激素（信号分子，如胰岛素），在体内进行化学反应的酶，以及抗病毒蛋白质，如干扰素。虽然非常不同，但它们都是在活细胞中产生的，活细胞中还含有数千种其他蛋白质。因此，它们需要被净化，否则污染物会毒害患者。一旦纯净，其脆弱的结构需要通过在可溶性和固体稳定剂存在下低温储存来稳定。这些生产阶段的开发称为生物加工，并依赖于敏感的技术来测量每个阶段产品的纯度，浓度和质量。我们已经有方法来观察蛋白质的3D结构，这些方法可以确保产品是正确的。然而，它们需要昂贵的仪器，大量的蛋白质和多年的经验才能有效地使用。我们已经使用这些方法，但希望开发对结构变化敏感的小型检测试剂盒，以便快速检测蛋白质质量的任何变化并进行适当的分析。这些试剂盒是基于缓慢，复杂的现有程序，目前需要太多的蛋白质是有用的。我们将开发从复杂混合物中特异性纯化目标蛋白的方法，以便可以分析该过程的更多阶段。我们还将减少传统检测所需的蛋白质量。然而，我们希望花大部分时间开发易于使用的套件，通过其他方式检测3D结构的变化。在一个例子中，我们将根据蛋白质与蜡状表面结合的强度来分离少量蛋白质。优质蛋白质不应该，而那些没有正确形成的蛋白质是粘性和结合的。另一个例子将使用一种化学物质，当它与蛋白质中未正确形成的部分结合时，它会发光。我们不仅打算验证这一点，而且还打算通过观察化学物质在什么温度下释放来测试蛋白质的稳定性。为了测试蛋白质是单个的还是聚集的，我们将使用小的化学接头来冷冻聚集状态，以便稍后根据蛋白质的大小进行分离的分析。最后，我们将使用一些小的酶来切割松软的蛋白质片段，以检测这些分子是正确折叠还是松散折叠。

Pharmaceuticals were once just small molecules such as aspirin and penicillin. These contain 21 and 42 atoms respectively and their structure is determined by the rigid bonds between each atom. Their purity is easily measured and any chemical changes are detected by established methods and their production is straight forward. Currently, a new generation of biopharmaceuticals is emerging which use large molecules such as those found in the body e.g. proteins, to selectively treat a range of disorders. One of the first biopharmaceuticals was insulin, from animal pancreas which since the 1920's has treated forms of diabetes caused by the patients' inability to make insulin. No small molecule can do what insulin does and now medicinal insulin is made by bacteria carrying the gene for human insulin. Insulin is a small protein but even so has 791 atoms linked not only by covalent bonds but also by weaker effects which stabilise its complex 3D structure. This exemplifies the main features of biopharmaceuticals; they offer highly selective effects of great medical value but are very complicated and difficult to make. Currently there is much discussion of drugs which treat previously untreatable conditions e.g. Herceptin and metastatic breast cancer. Herceptin is a protein called an antibody which kills specific cancer cells just as our own antibodies protect us from invading cells. Antibodies currently account for most new biopharmaceuticals and are complexes of more than 20,000 atoms. There are many other proteins used to target specific effects impossible to achieve with small molecules. These include hormones (signalling molecules like insulin), enzymes which carry out chemical reactions in the body, and antiviral proteins like interferons. Although very different, they all start off being made in living cells which also contain several thousand other proteins. Thus they need to be purified or contaminants will poison the patient. Once pure, their fragile structure needs to be stabilised by storage at low temperature in the presence of soluble and solid stabilising agents. Development of these stages of manufacture is called bio processing and relies upon sensitive techniques to measure the purity, concentration and quality of the product at every stage. We already have ways to look at the 3D structure of proteins and these are to ensure that the products are correct. However, they need expensive apparatus, large amounts of protein and years of experience to use effectively. We already use these methods but want to develop small test kits that are sensitive to structural changes so that any variation in protein quality can be rapidly detected and subjected to proper analysis. These kits are based upon slow, complex existing procedures that currently need too much protein to be useful. We will develop methods for the specific purification of target protein from complex mixtures so that more stages of the process can be analysed. We will also reduce the amounts of protein needed for the traditional assays. However, we wish to spend most time developing easy to use kits which detect changes in the 3D structure by other means. In one example we will separate small amounts of protein according to how much strongly they bind to a waxy surface. Good quality proteins should not whilst ones that have not properly formed are sticky and bind. Another example will use a chemical that lights up when it binds to parts of proteins that are not correctly formed. We intend not only to miniaturise this but also use it to test protein stability by observing at what temperature the chemical is released. To test whether the proteins are individual or aggregated we will use small chemical linkers to freeze the aggregated state for a later analysis which separates proteins according to their size. Finally, we will use small enzymes which cut floppy bits of protein to detect whether the molecules are correctly or loosely folded up.