Molecular Tools for Focused Proteomic Analysis of Bacterial Cell Factories
Fujii, Yu (2017)
Fujii, Yu
2017
Bioengineering
Teknis-luonnontieteellinen tiedekunta - Faculty of Natural Sciences
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Hyväksymispäivämäärä
2017-03-08
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tty-201703131153
https://urn.fi/URN:NBN:fi:tty-201703131153
Tiivistelmä
Cell factories are important and new tools to produce valuable products without depending on depleting fossil fuels. As there are successive cell factory constructions, there are emerging needs to develop better analysis tools for them. The conventional analysis methods such as 2-dimentional electrophoresis and mass spectrometry are certainly eligible for cell factory analysis, but they are not necessarily the most reasonable methods because of the need for special equipment. Therefore, new tools for cell factory analysis are needed.
Aldehyde-deformylating oxygenase (ADO) and bacterial luciferase (LuxAB) are interesting target proteins whose bacterial productions were analyzed here: ADO is an enzyme involved in fatty alkanes or alkenes generation in bacteria, and LuxAB is an enzyme catalyzing the bioluminescence production. Fatty alkanes and alkenes production by bacteria is drawing attention since it could replace the traditional chemical production of traffic fuels. Bioluminescence is also notable because it can be used as a real-time monitoring tool for certain bacterial production.
Therefore, in this study, immunoassay was studied as a new method for cell factory analysis. Especially, the immunoassay systems for ADO and LuxAB were constructed. The best working scFvs for the immunoassays were successfully selected, and the standard curves were also drawn for both of them. Thus, enzyme immunoassay using alkaline phosphatase (phoA) for ADO and a new immunoassay for LuxAB were established in this study. In the immunoassay for LuxAB, LuxAB generates the signals to be measured using exogenously added long-chain aldehyde as substrate, thus no tracer scFv or labels are needed. The systems were confirmed to work with ADO and LuxAB produced by Escherichia coli but not by Acinetobacter baylyi ADP1. The possible reason could be that A. baylyi ADP1 strains did not produce enough amounts of the proteins for the immunoassays. Thus, further research is still needed, but this study offers a meaningful foundation for the development of the immunoassays.
Aldehyde-deformylating oxygenase (ADO) and bacterial luciferase (LuxAB) are interesting target proteins whose bacterial productions were analyzed here: ADO is an enzyme involved in fatty alkanes or alkenes generation in bacteria, and LuxAB is an enzyme catalyzing the bioluminescence production. Fatty alkanes and alkenes production by bacteria is drawing attention since it could replace the traditional chemical production of traffic fuels. Bioluminescence is also notable because it can be used as a real-time monitoring tool for certain bacterial production.
Therefore, in this study, immunoassay was studied as a new method for cell factory analysis. Especially, the immunoassay systems for ADO and LuxAB were constructed. The best working scFvs for the immunoassays were successfully selected, and the standard curves were also drawn for both of them. Thus, enzyme immunoassay using alkaline phosphatase (phoA) for ADO and a new immunoassay for LuxAB were established in this study. In the immunoassay for LuxAB, LuxAB generates the signals to be measured using exogenously added long-chain aldehyde as substrate, thus no tracer scFv or labels are needed. The systems were confirmed to work with ADO and LuxAB produced by Escherichia coli but not by Acinetobacter baylyi ADP1. The possible reason could be that A. baylyi ADP1 strains did not produce enough amounts of the proteins for the immunoassays. Thus, further research is still needed, but this study offers a meaningful foundation for the development of the immunoassays.