Publikation:

Identification of Thermus aquaticus DNA polymerase variants with increased mismatch discrimination and reverse transcriptase activity from a smart enzyme mutant library

Lade...
Vorschaubild

Dateien

Raghunathan_2-6sc28hnon0yw8.pdf
Raghunathan_2-6sc28hnon0yw8.pdfGröße: 4.36 MBDownloads: 485

Datum

2019

Herausgeber:innen

Kontakt

ISSN der Zeitschrift

Electronic ISSN

ISBN

Bibliografische Daten

Verlag

Schriftenreihe

Auflagebezeichnung

ArXiv-ID

Internationale Patentnummer

Link zur Lizenz

Angaben zur Forschungsförderung

European Union (EU): 339834

Projekt

EvoEPIGEN - Evolved Replication Systems of Epigenetics
Open Access-Veröffentlichung
Open Access Gold
Core Facility der Universität Konstanz

Gesperrt bis

Titel in einer weiteren Sprache

Publikationstyp
Zeitschriftenartikel
Publikationsstatus
Published

Erschienen in

Scientific reports. 2019, 9, 590. eISSN 2045-2322. Available under: doi: 10.1038/s41598-018-37233-y

Zusammenfassung

DNA polymerases the key enzymes for several biotechnological applications. Obviously, nature has not evolved these enzymes to be compatible with applications in biotechnology. Thus, engineering of a natural scaffold of DNA polymerases may lead to enzymes improved for several applications. Here, we investigated a two-step approach for the design and construction of a combinatorial library of mutants of KlenTaq DNA polymerase. First, we selected amino acid sites for saturation mutagenesis that interact with the primer/template strands or are evolutionarily conserved. From this library, we identified mutations that little interfere with DNA polymerase activity. Next, these functionally active mutants were combined randomly to construct a second library with enriched sequence diversity. We reasoned that the combination of mutants that have minuscule effect on enzyme activity and thermostability, will result in entities that have an increased mutation load but still retain activity. Besides activity and thermostability, we screened the library for entities with two distinct properties. Indeed, we identified two different KlenTaq DNA polymerase variants that either exhibit increased mismatch extension discrimination or increased reverse transcription PCR activity, respectively.

Zusammenfassung in einer weiteren Sprache

Fachgebiet (DDC)
540 Chemie

Schlagwörter

Konferenz

Rezension
undefined / . - undefined, undefined

Forschungsvorhaben

Organisationseinheiten

Zeitschriftenheft

Zugehörige Datensätze in KOPS

Zitieren

ISO 690RAGHUNATHAN, Govindan, Andreas MARX, 2019. Identification of Thermus aquaticus DNA polymerase variants with increased mismatch discrimination and reverse transcriptase activity from a smart enzyme mutant library. In: Scientific reports. 2019, 9, 590. eISSN 2045-2322. Available under: doi: 10.1038/s41598-018-37233-y
BibTex
@article{Raghunathan2019-01-24Ident-44896,
  year={2019},
  doi={10.1038/s41598-018-37233-y},
  title={Identification of Thermus aquaticus DNA polymerase variants with increased mismatch discrimination and reverse transcriptase activity from a smart enzyme mutant library},
  volume={9},
  journal={Scientific reports},
  author={Raghunathan, Govindan and Marx, Andreas},
  note={Article Number: 590}
}
RDF
<rdf:RDF
    xmlns:dcterms="http://purl.org/dc/terms/"
    xmlns:dc="http://purl.org/dc/elements/1.1/"
    xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#"
    xmlns:bibo="http://purl.org/ontology/bibo/"
    xmlns:dspace="http://digital-repositories.org/ontologies/dspace/0.1.0#"
    xmlns:foaf="http://xmlns.com/foaf/0.1/"
    xmlns:void="http://rdfs.org/ns/void#"
    xmlns:xsd="http://www.w3.org/2001/XMLSchema#" > 
  <rdf:Description rdf:about="https://kops.uni-konstanz.de/server/rdf/resource/123456789/44896">
    <dcterms:abstract xml:lang="eng">DNA polymerases the key enzymes for several biotechnological applications. Obviously, nature has not evolved these enzymes to be compatible with applications in biotechnology. Thus, engineering of a natural scaffold of DNA polymerases may lead to enzymes improved for several applications. Here, we investigated a two-step approach for the design and construction of a combinatorial library of mutants of KlenTaq DNA polymerase. First, we selected amino acid sites for saturation mutagenesis that interact with the primer/template strands or are evolutionarily conserved. From this library, we identified mutations that little interfere with DNA polymerase activity. Next, these functionally active mutants were combined randomly to construct a second library with enriched sequence diversity. We reasoned that the combination of mutants that have minuscule effect on enzyme activity and thermostability, will result in entities that have an increased mutation load but still retain activity. Besides activity and thermostability, we screened the library for entities with two distinct properties. Indeed, we identified two different KlenTaq DNA polymerase variants that either exhibit increased mismatch extension discrimination or increased reverse transcription PCR activity, respectively.</dcterms:abstract>
    <dcterms:issued>2019-01-24</dcterms:issued>
    <dcterms:isPartOf rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/29"/>
    <dspace:isPartOfCollection rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/29"/>
    <dc:rights>Attribution 4.0 International</dc:rights>
    <dc:creator>Marx, Andreas</dc:creator>
    <dcterms:rights rdf:resource="http://creativecommons.org/licenses/by/4.0/"/>
    <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2019-02-07T10:19:41Z</dc:date>
    <void:sparqlEndpoint rdf:resource="http://localhost/fuseki/dspace/sparql"/>
    <foaf:homepage rdf:resource="http://localhost:8080/"/>
    <dcterms:available rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2019-02-07T10:19:41Z</dcterms:available>
    <dcterms:hasPart rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/44896/1/Raghunathan_2-6sc28hnon0yw8.pdf"/>
    <dc:creator>Raghunathan, Govindan</dc:creator>
    <bibo:uri rdf:resource="https://kops.uni-konstanz.de/handle/123456789/44896"/>
    <dc:contributor>Marx, Andreas</dc:contributor>
    <dc:contributor>Raghunathan, Govindan</dc:contributor>
    <dspace:hasBitstream rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/44896/1/Raghunathan_2-6sc28hnon0yw8.pdf"/>
    <dcterms:title>Identification of Thermus aquaticus DNA polymerase variants with increased mismatch discrimination and reverse transcriptase activity from a smart enzyme mutant library</dcterms:title>
    <dc:language>eng</dc:language>
  </rdf:Description>
</rdf:RDF>

Interner Vermerk

xmlui.Submission.submit.DescribeStep.inputForms.label.kops_note_fromSubmitter

Kontakt
URL der Originalveröffentl.

Prüfdatum der URL

Prüfungsdatum der Dissertation

Finanzierungsart

Kommentar zur Publikation

Allianzlizenz
Corresponding Authors der Uni Konstanz vorhanden
Internationale Co-Autor:innen
Universitätsbibliographie
Ja
Begutachtet
Ja
Diese Publikation teilen