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

用于蛋白质定向进化的三核苷酸交换方法的开发和演示

• 批准号: BB/E007384/1
• 负责人: Dafydd Jones
• 金额: $ 10.94万
• 依托单位: CARDIFF UNIVERSITY
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2006
• 资助国家: 英国
• 起止时间: 2006 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FE007384%2F1
• 关键词: Development; demonstration; trinucleotide; exchange; method

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.

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多数关键工作。蛋白质被合成为线性氨基酸序列，然后折叠形成其功能性3D结构。蛋白质达到其功能形状所需的所有信息都编码在线性氨基酸序列中，而线性氨基酸序列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效果，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性质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如何对蛋白质的特性做出贡献。它还使我们能够修改蛋白质的特性，以便在非自然环境中使用，例如用于工业应用。蛋白质的3D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性质的能力也是有限的。大自然采取了一种不同的、不那么理性的方法，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，因此拟议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以改变蛋白质的特性。我们将尝试改变一种称为TEM-1 β-内酰胺酶的蛋白质的特性，TEM-1 β-内酰胺酶是负责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的出现导致了新的和改进的青霉素类抗生素的开发。大自然很快做出了反应，TEM-1的变体很快进化出来，以克服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适应TEM-1来解毒这些新抗生素，帮助我们了解发生这种情况的自然过程。

项目成果

Structural and dynamic changes associated with beneficial engineered single-amino-acid deletion mutations in enhanced green fluorescent protein.

• DOI: 10.1107/s139900471401267x
• 发表时间: 2014-08
• 期刊: Acta crystallographica. Section D, Biological crystallography
• 作者: Arpino JA;Rizkallah PJ;Jones DD
• 通讯作者: Jones DD

Random single amino acid deletion sampling unveils structural tolerance and the benefits of helical registry shift on GFP folding and structure.

• DOI: 10.1016/j.str.2014.03.014
• 发表时间: 2014-06-10
• 期刊: Structure (London, England : 1993)
• 作者: Arpino JA;Reddington SC;Halliwell LM;Rizkallah PJ;Jones DD
• 通讯作者: Jones DD

In-frame amber stop codon replacement mutagenesis for the directed evolution of proteins containing non-canonical amino acids: identification of residues open to bio-orthogonal modification.

• DOI: 10.1371/journal.pone.0127504
• 发表时间: 2015
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Arpino JA;Baldwin AJ;McGarrity AR;Tippmann EM;Jones DD
• 通讯作者: Jones DD

Crystal structure of enhanced green fluorescent protein to 1.35 Å resolution reveals alternative conformations for Glu222.

• DOI: 10.1371/journal.pone.0047132
• 发表时间: 2012
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Arpino JA;Rizkallah PJ;Jones DD
• 通讯作者: Jones DD

Transposon-based approaches for generating novel molecular diversity during directed evolution.

基于转座子的方法，用于在定向进化过程中产生新的分子多样性。

• 发表时间: 2014
• 作者: []