Expanding the Scope of Protein Evolution

扩大蛋白质进化的范围

• 批准号: 6879140
• 负责人: DAVID R LIU
• 金额: $ 27.38万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6879140
• 关键词: DNA; biotechnology; cell death; endonuclease; enzyme activity; gene rearrangement; genetic manipulation; high throughput technology; microarray technology; molecular rearrangement; nucleic acid sequence; peptide chemical synthesis; polymerase chain reaction; protein engineering; recombinase; site directed mutagenesis; synthetic enzyme

Nature generates functional molecules through integrated cycles of diversification, selection, and amplification. While researchers have demonstrated the power of adapting this approach in the laboratory to generate proteins with desired binding or catalytic properties, the scope of protein molecular evolution is currently limited by the small number of strategies to screen or select for proteins with desired properties. Selections performed inside the living cell, in particular, are rare despite the significant advantages of in vivo selections over in vitro selections or screens. Moreover, while several proteins have been evolved to accept new substrates, the result of a selection or screen for new substrate tolerance is often an enzyme with broadened, rather than altered specificity. Our work seeks to address these existing limitations of protein molecular evolution. We propose the development of coupled positive and negative in vivo selections for the evolution of three classes of enzymes that manipulate biological macromolecules in powerful ways: recombinases, homing endonucleases, and inteins. The successful implementation of these methods will expand our understanding of these three important classes of enzymes and will validate the potential of coupled in vivo positive and negative selections to generate enzymes with truly altered, rather than simply broadened, specificities and activities. In addition, the successful evolution of each of these enzymes towards new activities may produce powerful research tools such as ligand-activated protein splicing domains and enzymes that specifically cleave or recombine DNA at sequences chosen by the researcher. Proteins evolved in this manner may also represent novel therapeutic approaches to protecting cells from viral infection and to manipulating virtually any protein-mediated process with a small molecule.

大自然通过多样化，选择和扩增的综合循环产生功能分子。 尽管研究人员已经证明了在实验室中适应这种方法以生成具有所需结合或催化特性的蛋白质的能力，但蛋白质分子进化的范围目前受到筛查或选择具有所需特性的蛋白质的少量策略的限制。 尽管体内选择比体外选择或筛选具有显着的优势，但在活细胞内进行的选择尤其是很少见。 此外，虽然已经进化了几种蛋白质以接受新的底物，但选择或屏幕的新底物耐受性的结果通常是具有扩展而不是改变特异性的酶。 我们的工作旨在解决蛋白质分子进化的现有局限性。 我们提出了三类酶的进化，以强大的方式操纵生物大分子：重物组织酶，归巢核酸内切蛋白和内部素的三类酶的进化，提出了耦合阳性和阴性的体内选择。 这些方法的成功实施将扩展我们对这三种重要类别酶类别的理解，并将验证体内正和负面选择中耦合的潜力，以生成具有真正变化的酶，而不是简单地扩展的特殊性和活动。 此外，这些酶中的每种酶的成功演变可能会产生强大的研究工具，例如配体激活的蛋白质剪接结构域和酶，这些酶是根据研究人员选择的序列特异性切割或重组DNA的酶。 以这种方式进化的蛋白质也可能代表了保护细胞免受病毒感染并几乎用小分子操纵任何蛋白质介导的过程的新型治疗方法。