Identification of Thermus aquaticus DNA polymerase variants with increased mismatch discrimination and reverse transcriptase activity from a smart enzyme mutant library
| Autor: | Govindan Raghunathan, Andreas Marx |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
DNA polymerase Mutant lcsh:Medicine DNA-Directed DNA Polymerase medicine.disease_cause Article Substrate Specificity 03 medical and health sciences 0302 clinical medicine Enzyme Stability medicine Genetic Testing Thermus lcsh:Science Saturated mutagenesis Thermostability Mutation Multidisciplinary biology Thermus aquaticus Chemistry lcsh:R RNA-Directed DNA Polymerase biology.organism_classification Reverse transcription polymerase chain reaction Kinetics 030104 developmental biology Biochemistry ddc:540 biology.protein lcsh:Q Mutant Proteins Primer (molecular biology) 030217 neurology & neurosurgery |
| Zdroj: | Scientific Reports Scientific Reports, Vol 9, Iss 1, Pp 1-14 (2019) |
| ISSN: | 2045-2322 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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