Large-scale preparation of biologically active recombinant chicken obese protein (leptin).
Prokaryotic expression vector pMON3401 encoding full size A(-1) chicken leptin (AF012727) was prepared by PCR of previously described cDNA. Escherichia coli cells transformed with this vector overexpressed large amounts of chicken leptin upon induction with nalidixic acid. The expressed protein found in the inclusion bodies was refolded and purified to homogeneity on a Q-Sepharose column, yielding two electrophoretically pure fractions (leptin-1 and leptin-2), eluted from the column by 100 and 125 mM NaCl. Both fractions showed a single band of the expected molecular mass of 16 kDa and were composed of over 95% of monomeric protein. The biological activity of both fractions, resulting from proper renaturation, was further evidenced by their ability to stimulate proliferation of leptin-sensitive BAF/3 cells transfected with a long form of human leptin-receptor construct and by lowering the food intake of starved chicken following intravenous or intraperitoneal injections.
Raver
N
N
Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, Rehovot, 76100, Israel.
Taouis
M
M
Dridi
S
S
Derouet
M
M
Simon
J
J
Robinzon
B
B
Djiane
J
J
Gertler
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001066:Appetite / Q000187:drug effects; D001402:B-Lymphocytes / Q000187:drug effects; D002352:Carrier Proteins / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D002645:Chickens / Q000378:metabolism*; D002853:Chromatography, Liquid; D018076:DNA, Complementary / Q000235:genetics; D003864:Depression, Chemical; D004926:Escherichia coli / Q000235:genetics; D005247:Feeding Behavior / Q000187:drug effects; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D020738:Leptin; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D017510:Protein Folding; D011506:Proteins / Q000008:administration & dosage / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014162:Transfection
D002352:Carrier Proteins; D018076:DNA, Complementary; C515448:LEPR protein, human; D020738:Leptin; D011506:Proteins; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; D011993:Recombinant Fusion Proteins; C515447:leptin receptor, mouse