Expression of a mammalian fatty acid-binding protein in Escherichia coli.
Rat liver fatty acid-binding protein (FABP) is a 14,200-Da polypeptide that is abundantly represented in the cytosol of liver and small intestinal epithelial cells. It may play an important role in the intracellular transport and metabolism of fatty acids. We have recently determined its sequence from an analysis of cloned cDNAs (Gordon, J.I., Alpers, D.H., Ockner, R. K., and Strauss, A.W. (1983) J. Biol. Chem. 258, 3356-3363). With the availability of full-length cloned FABP cDNAs, genetic manipulation of the coding region introduced into an appropriate expression vector could provide a powerful tool for analyzing the molecular details of fatty acyl-protein interaction. We, therefore, have inserted the coding sequence of rat FABP cDNA into a prokaryotic expression vector. Synthetic oligodeoxynucleotides were used to "properly" space the initiator methionine residue downstream from a Shine-Dalgarno ribosome-binding site. FABP transcription in the chimeric plasmid was under the direction of the leftward promotor of phage lambda. Promotor activity in turn was regulated by a thermolabile repressor specified by a defective lambda prophage contained in the host chromosome. When grown at permissive temperatures, rat liver FABP represented approximately 0.8% of radiolabeled soluble bacterial proteins. Edman degradation of FABP purified from Escherichia coli lysates indicated that it was intact. However, its NH2 terminus was not acetylated as it is in mammalian tissues. A frameshift mutation was introduced in vitro into the coding region of FABP cDNA. The effects of this on fatty acyl-protein interaction were examined using a solid phase (oleic acid-Sepharose) binding assay. Amino acid sequence rearrangements in the COOH-terminal region of rat liver FABP had a significant effect on its ability to bind fatty acids as well as on its stability in bacteria.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics*; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism*; D007421:Intestine, Small / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D010957:Plasmids; D051381:Rats
D002352:Carrier Proteins; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D004262:DNA Restriction Enzymes