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{"title":"Engineered Polymerases with Altered Substrate Specificity: Expression and Purification","authors":"Ali Nikoomanzar, Matthew R. Dunn, John C. Chaput","doi":"10.1002/cpnc.33","DOIUrl":null,"url":null,"abstract":"<p>Polymerase engineering is making it possible to synthesize xeno-nucleic acid polymers (XNAs) with diverse backbone structures and chemical functionality. The ability to copy genetic information back and forth between DNA and XNA has led to a new field of science known as synthetic genetics, which aims to study the genetic concepts of heredity and evolution in artificial genetic polymers. Since many of the polymerases needed to synthesize XNA polymers are not available commercially, researchers must express and purify these enzymes as recombinant proteins from <i>E. coli</i>. This unit details the steps needed to express, purify, and evaluate the activity of engineered polymerases with altered substrate recognition properties. The protocol requires 6 days to complete and will produce ∼20 mg of pure, nuclease-free polymerase per liter of <i>E. coli</i> bacterial culture. © 2017 by John Wiley & Sons, Inc.</p>","PeriodicalId":10966,"journal":{"name":"Current Protocols in Nucleic Acid Chemistry","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2017-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpnc.33","citationCount":"18","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current Protocols in Nucleic Acid Chemistry","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cpnc.33","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"Chemistry","Score":null,"Total":0}
引用次数: 18
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Abstract
Polymerase engineering is making it possible to synthesize xeno-nucleic acid polymers (XNAs) with diverse backbone structures and chemical functionality. The ability to copy genetic information back and forth between DNA and XNA has led to a new field of science known as synthetic genetics, which aims to study the genetic concepts of heredity and evolution in artificial genetic polymers. Since many of the polymerases needed to synthesize XNA polymers are not available commercially, researchers must express and purify these enzymes as recombinant proteins from E. coli . This unit details the steps needed to express, purify, and evaluate the activity of engineered polymerases with altered substrate recognition properties. The protocol requires 6 days to complete and will produce ∼20 mg of pure, nuclease-free polymerase per liter of E. coli bacterial culture. © 2017 by John Wiley & Sons, Inc.
改变底物特异性的工程聚合酶:表达和纯化
聚合酶工程使合成具有不同主链结构和化学功能的异种核酸聚合物(XNAs)成为可能。在DNA和XNA之间来回复制遗传信息的能力导致了一个被称为合成遗传学的新科学领域,其目的是研究人工遗传聚合物中的遗传和进化的遗传概念。由于合成XNA聚合物所需的许多聚合酶在商业上无法获得,研究人员必须从大肠杆菌中表达和纯化这些酶作为重组蛋白。本单元详细介绍了表达、纯化和评估具有改变底物识别特性的工程聚合酶活性所需的步骤。该方案需要6天完成,每升大肠杆菌培养物将产生~ 20mg纯无核酸酶聚合酶。©2017 by John Wiley &儿子,Inc。
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