Molecular Cloning, Expression, and Characterization of a Human Intestinal 15‐kDa Protein

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Abstract
We have isolated a cDNA encoding a human intestinal 15‐kDa protein (1–15P) from a human ileal λgt 11 cDNA library, using a full‐length rat I‐15P cDNA. One clone encompassed 571 nucleotides and encoded a 128–amino‐acid protein with a calculated molecular mass of 14355 Da. The deduced amino acid sequence of human I‐15P showed high similarity to the rat counterpart (78%), mouse ileal lipid‐binding protein (80%) and porcine gastrotropin (75%). It also exhibited 36% similarity to human liver fatty‐acid‐binding protein (L‐FABP). Northern blot analysis of human I‐15P revealed a single transcript only in ileum, however, the reverse‐transcription/PCR demonstrated expression in ovary and placenta, but it was much lower than in ileum. Transformation of Escherichia coli with the I‐15P cDNA resulted in the efficient expression of a protein that was identical to the ileal cytosolic 1–15P. In vitro binding studies revealed that the bacterially expressed recombinant I‐15P showed much lower affinities for palmitate and oleate than L‐FABP. However, it showed similar affinity for taurocholate, compared with a control, BSA. Comparison of the structural features of human I‐15P and human L‐FABP suggested that loss of a long α‐helix region and hydrophobic profile of I‐15P may be attributable to a unique ligand‐binding specificity of I‐15P.