Expression of a functionally active cardiac fatty acid-binding protein in the yeast, Saccharomyces cerevisiae.
The unicellular eukaryotic microorganism, Saccharomyces cerevisiae, transformed with a plasmid containing a cDNA fragment encoding bovine heart fatty acid-binding protein (H-FABP) under the control of the inducible yeast GAL10 promoter, expressed FABP during growth on galactose. The maximum level of immunoreactive FABP, identical in size to native protein as judged from SDS-polyacrylamide gel electrophoresis, was reached after approximately 16 hours of induction. Analysis of particulate and soluble subcellular fractions showed that FABP was exclusively associated with the cytosol. FABP expressed in yeast cells was functional as was demonstrated by its capacity to bind 14C-oleic acid in an in vitro assay. Growth of the transformants on galactose as the carbon source was significantly retarded at 37 degrees C. Whereas the fatty acid pattern of total lipids was not altered in transformed cells, desaturation of exogenously added 14C-palmitic acid was significantly reduced both at 30 and 37 degrees C. The lowest percentage of radioactively labeled unsaturated fatty acids was found in the phospholipid fraction.
Scholz
H
H
Institut für Biochemie und Lebensmittelchemie, Technische Universität Graz, Austria.
Kohlwein
S D
SD
Paltauf
F
F
Lezius
A
A
Spener
F
F
D002250:Carbon Radioisotopes; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000096:biosynthesis; D050556:Fatty Acid-Binding Proteins; D005690:Galactose / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D009206:Myocardium / Q000378:metabolism*; D009363:Neoplasm Proteins; D019301:Oleic Acid; D009829:Oleic Acids / Q000378:metabolism; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D014170:Transformation, Genetic
D002250:Carbon Radioisotopes; D002352:Carrier Proteins; D004271:DNA, Fungal; D050556:Fatty Acid-Binding Proteins; D009363:Neoplasm Proteins; D009829:Oleic Acids; D010169:Palmitic Acids; D019301:Oleic Acid; D019308:Palmitic Acid; D005690:Galactose