Linked Life Data

19763656

Source:http://linkedlifedata.com/resource/pubmed/id/19763656

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
10
pubmed:dateCreated
2010-1-29
pubmed:abstractText
Standard methods to quantify the activity of triacylglycerol (TAG) synthesizing enzymes DGAT and PDAT (TAG-SE) require a sensitive but rather arduous laboratory assay based on radio-labeled substrates. Here we describe two straightforward methods to detect TAG production in baker's yeast Saccharomyces cerevisiae. First we demonstrate that a quadruple knockout yeast strain deficient in storage lipids has a reduced growth rate in a medium supplemented with fatty acids. This phenotype is rescued by restoring TAG biosynthesis and can be thus used to select yeast cells expressing a recombinant TAG-SE. In the second method, the activity of the recombinant enzyme is measured in a fluorescent in situ assay using Nile red dye that is specific for neutral lipids. Correlation between Nile red fluorescence and enzyme activity is demonstrated with several mutants of a TAG synthesizing enzyme. This yeast live-cell-based assay is rapid, inexpensive, sensitive, and is amenable to high-throughput applications. The methods can be used for a variety of applications such as isolation of novel genes, directed evolution, gene-specific drug screening and will facilitate novel approaches in the research of TAG-SE.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Oct
pubmed:issn
1558-9307
pubmed:author
pubmed:issnType
Electronic
pubmed:volume
44
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
963-73
pubmed:meshHeading
pubmed:year
2009
pubmed:articleTitle
Simple methods to detect triacylglycerol biosynthesis in a yeast-based recombinant system.
pubmed:affiliation
Agricultural Lipid Biotechnology Program, Department of Agricultural, Food and Nutritional Science, University of Alberta, 4-10 Ag/For Centre, Edmonton, AB, T6G 2P5, Canada.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, Non-U.S. Gov't