High-throughput engineering of proteins: Sampling extended chemical diversity by combining directed evolution with an expanded genetic code.

每一种生物都含有数千种不同的蛋白质，它们执行维持生命所需的大部分关键工作。蛋白质也在工业中被用作治疗药物，而不是在正常的生物学背景下。蛋白质是由被称为氨基酸的化学物质的重复单位组成的分子。在绝大多数情况下，只有20种不同的氨基酸被用来组成单一蛋白质。这意味着蛋白质的化学性质仅限于从20种氨基酸中可获得的氨基酸。这反过来又限制了蛋白质可以采样的可用功能。扩大蛋白质可用的化学类型将允许引入目前在自然界中不可用的新性质，这将为研究生物系统和使蛋白质适应生物技术应用开辟新的途径。因此，我们假设，通过掺入非天然氨基酸来扩大蛋白质样本的化学多样性可以为蛋白质引入新的化学成分，从而产生具有新颖和增强性质的变体。组成蛋白质的氨基酸序列通过基因在遗传物质中编码。在细胞内的蛋白质中加入非天然氨基酸的一种方法是通过改变组成基因的单个密码子来改变基因的编码特性(密码子准确地编码哪些氨基酸将被添加到不断增长的蛋白质链中)。这可以通过改变或改造细胞蛋白质合成机制的组件来实现，以便将非天然氨基酸结合到不断增长的蛋白质链中，以响应特定的密码子。在这个项目中，我们提出了一种方法，将任何数量的新的非天然氨基酸基因编码成任何蛋白质。我们的方法以高通量的方式瞄准蛋白质的整个序列，从而潜在地检查每一个可能的氨基酸位置对蛋白质性质的影响。这种方法被称为定向进化，是一种基于达尔文进化论的强大而成功的策略，可以改变蛋白质的性质，但它针对单个基因，并在试管中以受控方式发生。我们提出的方法将首次证明人工蛋白质进化可以涉及非天然合成氨基酸的使用。由于我们对在蛋白质的特定氨基酸位置加入非天然氨基酸的影响的了解是有限的，因此有一种方法可以快速对蛋白质中的许多不同位置进行采样将是非常有用的。这反过来将使我们能够通过采样新的化学功能来充分开发构建具有新的有用性质的蛋白质的潜力。

Every living thing contains thousands of different proteins that perform most of the crucial jobs needed to maintain life. Proteins are also being utilised outside of their normal biological context as therapeutics in industry. Proteins are molecules that consist of repeating units of chemicals called amino acids. In the vast majority of cases, only 20 different amino acids are used to compose a single protein. This means that the chemical nature of proteins is restricted to what is available from the 20 amino acids. This in turn limits the available functionality that a protein can sample. Expanding the type of chemistry available to a protein will allow new properties not currently available in nature to be introduced, which will open up new routes to the study of biological systems and for adapting proteins for biotechnological applications. Therefore, we hypothesise that expanding the chemical diversity sampled by a protein by the incorporation of unnatural amino acids can introduce new chemistry into a protein so generating variants with novel and enhanced properties. The sequence of amino acids that comprise a protein is encoded in the genetic material via a gene. One approach for incorporation of unnatural amino acids into a protein within a cell is to alter the coding properties of a gene via changes to the individual codons comprising the gene (codons encode precisely which amino acid will be added to the growing protein chain). This can be achieved by altering or engineering components of the cell protein synthesising machinery so that the unnatural amino acid is incorporated into the growing protein chain in response to a specific codon. In this project, we present a method to genetically encode any number of novel unnatural amino acids into any protein. Our method targets the entire sequence of a protein in a high-throughput manner such that potentially every possible amino acid position is examined for its effects on the properties of a protein. This approach, termed directed evolution, is a powerful and successful strategy based on Darwinian evolution to alter the properties of proteins but is targeted to an individual gene and takes place in a controlled fashion in the test-tube. Our proposed method will be the first to demonstrate that artificial protein evolution can involve the use of unnatural synthetic amino acids. Since our knowledge of the effect of incorporating an unnatural amino acid at particular amino acid position in a protein is limited, having a method that can quickly sample many different positions throughout a protein would be extremely useful. This will in turn allow us to fully exploit the potential of constructing proteins with new and useful properties through in the sampling of new chemical functionalities.