Mutant firefly luciferases with improved specific activity and dATP discrimination constructed by yeast cell surface engineering
| Autor: | Tatsuya Fushimi, Kouichi Kuroda, Mitsuyoshi Ueda, Hideya Shintani, Hiroyuki Tsunoda, Natsuko Miura |
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| Rok vydání: | 2012 |
| Předmět: |
chemistry.chemical_classification
Luciferases biology Mutant General Medicine Protein Engineering biology.organism_classification Applied Microbiology and Biotechnology Molecular biology Yeast DNA sequencing Substrate Specificity Amino acid Deoxyadenine Nucleotides Enzyme Biochemistry chemistry Luciferases Firefly Yeasts Photinus pyralis heterocyclic compounds Luciferase Biotechnology |
| Zdroj: | Applied Microbiology and Biotechnology. 97:4003-4011 |
| ISSN: | 1432-0614 0175-7598 |
| Popis: | Pyrosequencing system utilizing luciferase is one of the next-generation DNA sequencing systems. However, there is a crucial problem with the current pyrosequencing system: luciferase cannot discriminate between ATP and dATP completely, and dATPαS must be used as the dATP analogue. dATPαS is expensive and has low activity for the enzyme. If luciferase can clearly recognize the difference between ATP and dATP, dATP could be used instead of the expensive dATPαS in the pyrosequencing system. We attempted to prepare a novel luciferase with improved specific activity and dATP discrimination with the molecular display method. First, we selected two amino acid residues, Ser440 and Ser456, as target residues for mutation from the whole sequence of Photinus pyralis luciferase; we comprehensively mutated these two amino acids. A mutant luciferase library was constructed using yeast cell surface engineering. Through three step-wide screenings with individual conditions, we easily and speedily isolated three candidate mutants from 1,152 candidates and analyzed the properties of these mutants. Consequently, we succeeded in obtaining interesting mutant luciferases with improved specific activity and dATP discrimination more conveniently than with other methods. |
| Databáze: | OpenAIRE |
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