Development and demonstration of a trinucleotide exchange method for the directed evolution of proteins

每一种生物都含有数千种不同的蛋白质，这些蛋白质执行维持生命所需的大部分关键工作。蛋白质被合成为氨基酸的线性序列，然后折叠形成它们的功能三维结构。蛋白质达到其功能形状所需的所有信息都编码在氨基酸的线性序列中，而氨基酸又由该蛋白质基因的DNA序列编码。进化的过程包括通过改变基因来改变蛋白质的氨基酸序列。因此，这些变化可以改变蛋白质的特性，并可能转化为有益的影响，使生物体能够在特定的环境挑战中生存下来。蛋白质氨基酸序列的变化如何转化为蛋白质特性的变化是生物学中最基本的问题之一。基因工程的出现使我们能够随意改变基因的DNA序列，从而改变蛋白质中氨基酸线性序列的性质。这使我们能够了解某些氨基酸对蛋白质特性的影响。它还允许我们修改蛋白质的特性，以便在非自然环境中使用，例如工业应用。蛋白质的三维结构非常复杂，我们对它的理解仍然有限。因此，我们预测特定设计的突变如何影响蛋白质特性的能力也是有限的。大自然采取了一种不同的、不那么理性的方式，它随机引入突变，只选择那些有益的突变。由于自然界已经非常成功地使蛋白质适应多种功能，因此提出的研究旨在通过开发一种方法，在实验室中复制进化过程，该方法可以将突变随机引入基因中，以改变蛋白质的特性。我们将尝试修改一种叫做TEM-1 β -内酰胺酶的蛋白质的特性，这种蛋白质是对青霉素等抗生素产生耐药性的蛋白质之一。细菌对传统抗生素的耐药性导致了新的和改进的类青霉素抗生素的发展。大自然很快做出了反应，TEM-1的变体很快就进化出来，克服了这些新抗生素的毒性作用。使用我们的新方法，该项目将研究如何以及哪些突变有助于使TEM-1适应这些新抗生素的解毒，帮助我们了解这种情况发生的自然过程。

Every living thing contains thousands of different proteins that carry out most of the crucial jobs needed to maintain life. Proteins are synthesised as a linear sequence of amino acids, which then fold to form their functional 3D structures. All the information required for a protein to reach its functional shape is encoded in the linear sequence of amino acids, which in turn is encoded by the DNA sequence of the gene for that protein. The process of evolution involves changes to the amino acid sequence of a protein through changes to the gene. As a consequence, these changes can alter the properties of the protein and may translate into beneficial effects, allowing the organism to survive a particular environmental challenge. How changes to the amino acid sequence of a protein translate into changes in the properties of a protein is one of the most fundamental questions in biology. The advent of genetic engineering has enabled us to change the DNA sequence of genes at will and so change the nature of the linear sequence of amino acids in the protein. This has allowed us to understand how certain amino acids contribute towards the properties of a protein. It has also allowed us to modify the characteristics of proteins for use in unnatural environments such as for industrial applications. The 3D structure of a protein is highly complex and our understanding of it is still limited. Therefore our ability to predict how a particular designed mutation will affect the properties of the protein is also limited. Nature takes a different, less rational approach, by introducing mutations at random and selecting only those mutations that are of benefit. As nature has already been very successful in adapting proteins for a multitude of functions, the proposed research aims to copy the process of evolution in the laboratory by developing a method that can introduce mutations randomly into a gene in order to change the properties of a protein. We will attempt to modify the characteristics of a protein called TEM-1 beta-lactamase, one of the proteins responsible for resistance to antibiotics such as penicillin. The advent of bacterial resistance to the traditional antibiotics led to the development of new and improved penicillin-like antibiotics. Nature was quick to respond and variants of TEM-1 soon evolved to overcome the toxic effects of these new antibiotics. Using our new method, the project will investigate how and which mutations contribute to adapting TEM-1 to detoxify these new antibiotics, helping us to understand the natural process by which this occurs.