Molecular cloning, expression, and characterization of a human intestinal 15-kDa protein.
We have isolated a cDNA encoding a human intestinal 15-kDa protein (I-15P) from a human ileal lambda gt 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 I-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 alpha-helix region and hydrophobic profile of I-15P may be attributable to a unique ligand-binding specificity of I-15P.
Fujita
M
M
Department of Biochemistry, Niigata University School of Medicine, Japan.
Fujii
H
H
Kanda
T
T
Sato
E
E
Hatakeyama
K
K
Ono
T
T
D000368:Aged; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001647:Bile Acids and Salts / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000302:isolation & purification; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism; D005768:Gastrointestinal Hormones; D006801:Humans; D007082:Ileum / Q000737:chemistry*; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018996:Myelin P2 Protein / Q000737:chemistry; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification; D025521:Tumor Suppressor Proteins
D001647:Bile Acids and Salts; D002352:Carrier Proteins; D018076:DNA, Complementary; C493319:FABP1 protein, human; C107608:FABP7 protein, human; C493320:Fabp1 protein, mouse; C493321:Fabp1 protein, rat; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D005768:Gastrointestinal Hormones; D018996:Myelin P2 Protein; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; C057275:fatty acid-binding protein 6