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.

Carbon Radioisotopes↗

PubMedDate not supplied

Expression of fatty acid-binding protein from bovine heart in Escherichia coli.

The coding part of the cDNA of cardiac fatty acid-binding protein (cFABP) from bovine heart was cloned into the vector pKK233-2. After induction with isopropyl-beta-D-thiogalactopyranoside cFABP was found in a soluble form in the cytosol of plasmid transformed E. coli amounting up to 5.7% of the soluble protein. cFABP was detected after SDS-polyacrylamide gelelectrophoresis and/or isoelectric focusing and Western blot by immuno-staining and was determined quantitatively by a solid phase enzyme-linked immuno sorbent assay. The cFABP produced by bacteria binds oleic acid with high affinity as shown by comigration of protein and ligand in both gelfiltration and isoelectric focusing. cFABP was purified from bacterial lysates to near homogeneity and resolved into four isoproteins.

Animals↗