Document Detail

Addition of an N-terminal epitope tag significantly increases the activity of plant fatty acid desaturases expressed in yeast cells.
MedLine Citation:
PMID:  19137289     Owner:  NLM     Status:  MEDLINE    
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.
Jami B O'Quin; Robert T Mullen; John M Dyer
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2009-01-10
Journal Detail:
Title:  Applied microbiology and biotechnology     Volume:  83     ISSN:  1432-0614     ISO Abbreviation:  Appl. Microbiol. Biotechnol.     Publication Date:  2009 May 
Date Detail:
Created Date:  2009-04-21     Completed Date:  2009-05-11     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  8406612     Medline TA:  Appl Microbiol Biotechnol     Country:  Germany    
Other Details:
Languages:  eng     Pagination:  117-25     Citation Subset:  IM    
United States Department of Agriculture, Agricultural Research Service, Southern Regional Research Center, 1100 Robert E. Lee Blvd., New Orleans, LA, USA.
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MeSH Terms
Amino Acid Sequence
Base Sequence
Fatty Acid Desaturases / genetics,  metabolism*
Gene Expression*
Genes, myc / genetics
Hemagglutinins / genetics
Molecular Sequence Data
Plant Proteins / genetics,  metabolism*
Recombinant Fusion Proteins / genetics,  metabolism
Saccharomyces cerevisiae / enzymology*,  genetics*
Reg. No./Substance:
0/Hemagglutinins; 0/Plant Proteins; 0/Recombinant Fusion Proteins; EC 1.14.19.-/Fatty Acid Desaturases

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