Entirely in vivo enzyme evolution and engineering of Cre recombinase
Cre 重组酶的全体内酶进化和工程
基本信息
- 批准号:8011691
- 负责人:
- 金额:$ 5.05万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2009
- 资助国家:美国
- 起止时间:2009-07-01 至 2011-06-30
- 项目状态:已结题
- 来源:
- 关键词:AddressArtificial OrgansBiological AssayBiological ModelsCellsCollectionComplexDNADevelopmentEffectivenessEngineeringEnzymesEvolutionGene TargetingGenerationsGenesGeneticGoalsHealthIn VitroLibrariesMethodologyMethodsModelingMusMutagenesisMutationNonsense MutationPartner in relationshipPathway interactionsPharmaceutical PreparationsPharmacologic SubstancePlasmidsPopulationProceduresProcessPropertyProtein EngineeringProteinsProtocols documentationReportingReproductionResearchSaccharomyces cerevisiaeSaccharomycetalesSiteSpecificitySpeedSubstrate SpecificitySystemTechniquesTechnologyTestingTherapeuticVariantVirtual LibraryYeastsbasedaughter celldirected evolutionendonucleasehomologous recombinationimprovedin vivointerestmutantnovelnucleasepressurepublic health relevancerecombinaseremediationrepairedresearch studysuccesstrend
项目摘要
DESCRIPTION (provided by applicant): Directed evolution is a versatile method for the creation of proteins with novel properties. The evolution of enzymes has already provided variants with higher stability, increased efficiency, and altered specificity or enantioselectivity. As the trend of using enzymes in industrial processes, the synthesis of pharmaceuticals, and consumer health and cleaning products continues to grow, newer and better directed evolution techniques will be required to solve more complex problems. One major limitation of current directed evolution approaches is the random library used to search for improved proteins. Current technology enables average libraries on the order of 10'^8-10'^9 variants, while research has suggested that much larger libraries (>10'^13) will be necessary to discover enzymes with significantly altered specificities or entirely new functions. This proposal describes a new system in yeast that aims to overcome current limitations on library size. Aim 1 describes a method for performing in vivo mutagenesis of a target gene within the budding yeast S. cerevisiae. This method relies on the highly efficient homologous recombination machinery in yeast. Mutagenic libraries, introduced on plasmids, will be liberated using a highly specific endonuclease. The resulting linear DNA strands will target and mutagenize a gene of interest through homologous recombination. Aim 2 outlines a method for sharing mutagenic libraries between yeast to increase potential library size. By allowing two collections of yeast with complementary random libraries to mate, those two libraries can be shared among the entire population. A selective pressure will be applied, thus propagating only the best variants while essentially searching all possible mutants. Such a protocol has the potential to search very large virtual libraries well beyond the ability of existing technology. Aim 3 proposes a directed evolution experiment that uses the methods developed in Aims 1 and 2. A new variant of Cre, a site-specific recombinase widely used in mouse genetics, will be evolved using the in vivo mutagenesis and mating procedures. This experiment should convincingly demonstrate the utility of these new methods. PUBLIC HEALTH RELEVANCE: This proposal describes a new technique for directed evolution of enzymes. New enzymes generated using this technique could be used in the discovery and synthesis of new drugs or as drugs themselves. The proposed methodology could provide access to proteins with uses as varied as cleaning products, environmental remediation, and artificial organs.
描述(由申请人提供):定向进化是一种用于创造具有新特性的蛋白质的通用方法。酶的进化已经提供了更高的稳定性、更高的效率和改变的特异性或对映体选择性的变体。随着在工业过程、药物合成以及消费者健康和清洁产品中使用酶的趋势持续增长,将需要更新和更好的定向进化技术来解决更复杂的问题。当前定向进化方法的一个主要限制是用于搜索改进的蛋白质的随机文库。目前的技术能够使平均文库达到10‘^8-10’^9个变异体的数量级,而研究表明,需要更大的文库(>;10‘^13)来发现具有显著改变的特异性或全新功能的酶。这项建议描述了一种新的酵母系统,旨在克服目前对文库大小的限制。目的1描述了一种在萌芽酵母中对目标基因进行体内诱变的方法。这种方法依赖于酵母中高效的同源重组机制。利用一种高度特异的内切酶,可以释放通过质粒引入的致突变文库。由此产生的线性DNA链将通过同源重组来靶向和突变感兴趣的基因。目的2概述了一种在酵母之间共享突变文库以增加潜在文库大小的方法。通过允许具有互补随机文库的两个酵母集合交配,这两个文库可以在整个种群中共享。将施加选择性压力,从而只繁殖最好的变异,而实质上搜索所有可能的突变。这样的协议有可能搜索非常大的虚拟图书馆,远远超出现有技术的能力。AIM 3提出了一个定向进化实验,它使用了AIMS 1和AIMS 2中开发的方法。Cre是一种广泛应用于小鼠遗传学的位点特异性重组酶,将通过体内突变和交配过程进化出一种新的变种。这个实验应该能令人信服地证明这些新方法的实用性。与公共卫生相关:这项提案描述了一种酶的定向进化的新技术。利用这项技术产生的新酶可以用于新药的发现和合成,也可以用作药物本身。拟议的方法可以提供获取蛋白质的途径,其用途广泛,包括清洁产品、环境修复和人造器官。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Dante William Romanini其他文献
Dante William Romanini的其他文献
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{{ truncateString('Dante William Romanini', 18)}}的其他基金
Entirely in vivo enzyme evolution and engineering of Cre recombinase
Cre 重组酶的全体内酶进化和工程
- 批准号:
7751461 - 财政年份:2009
- 资助金额:
$ 5.05万 - 项目类别:
Entirely in vivo enzyme evolution and engineering of Cre recombinase
Cre 重组酶的全体内酶进化和工程
- 批准号:
8306461 - 财政年份:2009
- 资助金额:
$ 5.05万 - 项目类别:
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