Molecular cloning of the obese gene from Cyprinus carpio and its expression in Escherichia coli

Abstract

Aiming to analyze the characteristics of the Cyprinus carpio obese gene structure and the biological activity of its expression product, we amplified the carp obese gene using reverse transcription polymerase chain reaction from carp mesentery adipose tissue RNA. Sequence analysis revealed that it has a length of 438 nt, which encodes a 146-amino acid peptide. When nucleotide sequence and deduced amino acid sequence were compared with homologous sequences from those of humans, pigs, and rats, they displayed a fairly high degree of conservation (the homology of the nucleotide sequence was 84%, 86%, and 95%, respectively, while that of the amino acid sequence was 84%, 82%, and 96%, respectively, for humans, pigs, and rats). The cDNA fragment was inserted into the expression vector pET-28a, and the resulting plasmid was expressed in Escherichia coli BL21 (DE3) by isopropylthiogalactoside induction. Results of sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis indicated that a fusion protein was specifically expressed in E. coli BL21 (DE3). The weight of the fusion protein was about 20 kDa, and a 16-kDa protein was expressed from the carp obese gene. By gel thin-layer scanning analysis, the amount of target protein was determined to be about 20%. The purified product was found to be biologically active and to reduce the food intake and body weight of mice during tests.