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Published on: June 1, 2014
Mutant firefly luciferases with improved specific activity and dATP discrimination constructed by yeast cell surface
Tatsuya Fushimi1, Natsuko Miura, Hideya Shintani
1Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Applied Microbiology and Biotechnology
|November 15, 2012
Summary
Researchers engineered a novel luciferase enzyme for DNA sequencing. This improved enzyme better distinguishes between ATP and dATP, potentially reducing costs and increasing efficiency in pyrosequencing systems.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Pyrosequencing is a next-generation DNA sequencing technology.
- Current systems use luciferase, which struggles to differentiate between ATP and dATP.
- This necessitates the use of expensive dATPαS, an analogue with lower enzyme activity.
Purpose of the Study:
- To engineer a novel luciferase with enhanced specific activity and improved dATP discrimination.
- To enable the direct use of dATP instead of dATPαS in pyrosequencing.
Main Methods:
- Utilized molecular display and yeast cell surface engineering to create a mutant luciferase library.
- Focused mutations on Ser440 and Ser456 residues of Photinus pyralis luciferase.
- Employed a three-step screening process to isolate promising mutants from 1,152 candidates.
Main Results:
- Successfully isolated three candidate mutant luciferases.
- These mutants exhibited improved specific activity and enhanced dATP discrimination.
- The engineering process was more convenient and efficient than previous methods.
Conclusions:
- Developed novel mutant luciferases with superior performance for pyrosequencing.
- Demonstrated a more efficient method for enzyme engineering.
- The findings pave the way for cost-effective and improved DNA sequencing.
