THE RABBIT ILEAL LIPID-BINDING PROTEIN - GENE CLONING AND FUNCTIONAL EXPRESSION OF THE RECOMBINANT PROTEIN

A bile-acid-binding protein of M(r) 14000 has been previously identifi ed by photoaffinity labeling in rabbit ileal brush border membrane ves icles [Kramer et al. (1993) J. Biol. Chem. 268, 18035-18046]. This per ipheral membrane associated protein was purified and identified as an ileal lipid-binding protein. It was further shown to be identical to t he cytosolic 14-kDa bile-acid-binding protein from the same tissue. St arting with sequence information from tryptic fragments, we cloned and sequenced the gene and its transcript. It has four exons (123, 176, 9 0, 115 bp) and three introns (1372, 2291, 3137 bp) and a similar struc ture as the genes from other members of the fatty-acid-binding protein family. The deduced protein has 128 amino acid residues and a calcula ted molecular mass of 14404 Da. It exhibits high similarity to its hum an (83%), mouse (77%), rat (76%) and porcine (72%) counterparts. Furth ermore, the recombinant protein was produced in Escherichia coli and s hown to be identical to native protein from ileal tissue. Functionalit y of the recombinant protein was demonstrated by labeling with various photoaffinity derivatives of bile acids. Ranking of the photolabeling efficiency of these probes towards the recombinant protein was compar able to the respective ranking towards the native protein. Polyclonal antibodies that were raised in hens against the recombinant protein, s pecifically recognized the ileal lipid-binding protein in the brush bo rder membrane and cytosol from rabbit ileum, In contrast. no labeling was observed with jejunal tissue. Our results suggest a specific role of the membrane-associated ileal lipid-binding protein for the process of ileal bile acid uptake.