NDLI: Addition of an N-terminal epitope tag significantly increases the activity of plant fatty acid desaturases expressed in yeast cells

Content Provider	Springer Nature Link
Author	O’Quin, Jami B. Mullen, Robert T. Dyer, John M.
Copyright Year	2009
Abstract	Saccharomyces cerevisiae shows great potential for development of bioreactor systems geared toward the production of high-value lipids such as polyunsaturated omega-3 fatty acids, the yields of which are largely dependent on the activity of ectopically expressed enzymes. Here, we show that the addition of an N-terminal epitope tag sequence (either Myc or hemagglutinin) to oleate desaturase (FAD2) or omega-3 linoleate desaturase (FAD3) enzymes from plants, which catalyze consecutive reactions in the production of long chain omega-3 fatty acids, significantly increases their activity up to fourfold when expressed in yeast cells. Quantitative protein blotting using an antibody specific for native FAD2 revealed that the steady-state amount of the epitope-tagged FAD2 protein was also approximately fourfold higher than that of its untagged counterpart, demonstrating a direct relationship between the epitope tag-induced increase in enzyme amount and fatty acid product formation. Protein half-life and RNA blotting experiments indicated that the half-lives and mRNA content of the tagged and untagged FAD2 proteins were essentially the same, suggesting that the epitope tags increased protein abundance by improving translational efficiency. Taken together, these results indicate that the addition of an epitope tag sequence to a plant fatty acid desaturase (FAD) not only provides a useful means for protein immunodetection using highly specific, commercially available antibodies, but that it also significantly increases FAD activity and the production of polyunsaturated fatty acids in yeast cells.
Starting Page	117
Ending Page	125
Page Count	9
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	83
Issue Number	1
e-ISSN	14320614
Language	English
Publisher	Springer-Verlag
Publisher Date	2009-01-10
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	Epitope tag Fatty acid desaturase Lipids Omega-3 fatty acids Saccharomyces cerevisiae Microbial Genetics and Genomics Microbiology Biotechnology
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

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