Recombinant expression of biologically active rat leptin in Escherichia coli.
Leptin is a 16-kDa nonglycosylated hormone that is produced in mature adipocytes and which acts primarily in the hypothalamus to reduce food intake and body weight. While the rat is a representative laboratory animal model in obesity research, so far recombinant rat leptin was not available. In the present study, rat leptin was recombinantly expressed in Escherichia coli and purified in a bioactive form to provide a further tool for the analysis of leptin functions in rats. Leptin cDNA was cloned by RT-PCR from total RNA of SD rat adipocytes, and overexpression was achieved by subcloning the leptin cDNA into the pET-29a vector, which enabled the recombinant expression of rat leptin as an S-peptide-tagged fusion protein. Since the fusion proteins were expressed in inclusion bodies, after purification of the insoluble fraction, leptin proteins were refolded by sequential dialysis into physiological buffers. The biological activity of this recombinant protein was confirmed in proliferation assays using leptin-sensitive rat insulinoma cells as well as a newly developed leptin-sensitive luciferase assay system. The specific binding of the S-tagged leptin to leptin-receptor-expressing cells was further shown by flow cytometry using fluorescence-conjugated S-proteins.
Park
J H
JH
Protein Engineering Laboratory, Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejon 305-600, Korea.
Lee
H H
HH
Na
S Y
SY
Ju
S K
SK
Lee
Y J
YJ
Lee
M K
MK
Kim
K L
KL
D017667:Adipocytes / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002455:Cell Division; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D007340:Insulinoma / Q000473:pathology; D020738:Leptin / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D017510:Protein Folding; D020673:Protein Renaturation; D051381:Rats; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013917:Thrombin / Q000378:metabolism; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D018076:DNA, Complementary; C515448:LEPR protein, human; D020738:Leptin; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; D011993:Recombinant Fusion Proteins; D013917:Thrombin