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Cloning of Chinese obese cDNA and its expression in E. coli
1Research Institute of Pediatrics, Beijing Children's Hospital, Capital University of Medical Sciences, Beijing 100045, China.
Objective:
To obtain the sequence of Chinese obese (OB) cDNA and establish a method of leptin production in China.
Methods:
Han Chinese OB cDNA fragment was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) with total RNA extracted from human adipocytes and was inserted into the expressing vector pBV220. Then the constructed recombinant plasmid pBV220-OB was transformed to E. coli DH5 alpha for leptin expression. The recombinant expressing system was confirmed by restriction endonuclease digestion, DNA sequencing and protein expression. E. coli cells were lysed by high-pressure homogenization. After cell membrane was extracted, the inclusion bodies were mainly renatured and purified primarily by precipitation with ammonium sulfate and gel chromatography through a Sephadex G75 column. The activity of recombinant leptin was determined by its influence on the satiety and weight gain of mice.
Results:
Analysis of DNA sequence showed that Han Chinese OB cDNA included the glutamine codon at 49. The amount of recombinant leptin expressed in E. coli accounted for 31%-47% of total cellular proteins. From 1 L of fermentative bacteria about 40 mg of pure recombinant human leptin was isolated with a purity of being above 95%. The recombinant human leptin could reduce food intake and inhibit weight gains in mice.
Conclusion:
The glutamine codon at 49 is not missing in Chinese OB gene. The biologically active human leptin can be obtained by a relatively simple method of recombinant DNA technology.