Engineering TaqII bifunctional endonuclease DNA recognition fidelity: the effect of a single amino acid substitution within the methyltransferase catalytic site
Autor: | Weronika Wrese, Joanna Zebrowska, Ewa Sulecka, Agnieszka Zylicz-Stachula, Piotr M. Skowron, Edyta Czajkowska |
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Rok vydání: | 2015 |
Předmět: |
0301 basic medicine
Methyltransferase medicine.disease_cause 03 medical and health sciences Endonuclease Sinefungin Catalytic Domain Genetics medicine Escherichia coli Cloning Molecular Thermus Deoxyribonucleases Type II Site-Specific Molecular Biology Gene chemistry.chemical_classification biology General Medicine Protein engineering Molecular biology Restriction enzyme 030104 developmental biology Enzyme chemistry Biochemistry Amino Acid Substitution biology.protein Mutagenesis Site-Directed |
Zdroj: | Molecular biology reports. 43(4) |
ISSN: | 1573-4978 |
Popis: | The aim of this study was to improve a useful molecular tool-TaqII restriction endonuclease-methyltransferase-by rational protein engineering, as well as to show an application of our novel method of restriction endonuclease activity modulation through a single amino acid change in the NPPY motif of methyltransferase. An amino acid change was introduced using site-directed mutagenesis into the taqIIRM gene. The mutated gene was expressed in Escherichia coli. The protein variant was purified and characterized. Previously, we described a TspGWI variant with an amino acid change in the methyltransferase motif IV. Here, we investigate a complex, pleiotropic effect of an analogous amino acid change on its homologue-TaqII. The methyltransferase activity is reduced, but not abolished, while TaqII restriction endonuclease can be reactivated by sinefungin, with an increased DNA recognition fidelity. The general method for engineering of the IIS/IIC/IIG restriction endonuclease activity/fidelity is developed along with the generation of an improved TaqII enzyme for biotechnological applications. A successful application of our novel strategy for restriction endonuclease activity/fidelity alteration, based on bioinformatics analyses, mutagenesis and the use of cofactor-analogue activity modulation, is presented. |
Databáze: | OpenAIRE |
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