Purification and characterization of secreted human leptin produced in baculovirus-infected insect cells.
The product of the human ob (obesity) gene, leptin, appears to function in the maintenance of body weight in vivo. When injected into mice, this hormone reduces food consumption and causes weight loss. This work has been done with recombinant leptin (re-leptin) purified and renatured from inclusion bodies in Escherichia coli. We have expressed the human obesity gene encoding the predicted full-length leptin in Spodoptera frugiperda (Sf-9) cells by infection with the recombinant baculovirus system. Protein corresponding to re-leptin was secreted into the culture medium and purified in sufficient quantity for testing biological activity. The secreted re-protein was characterized and found to be unmodified except for correct cleavage of the signal peptide during export from the cells. The resulting molecule is expected to be properly folded and has been purified to a high level of homogeneity. The re-leptin secreted from Sf-9 cells should be an appropriate source of protein for study of the native structure.
Churgay
L M
LM
Department of Biotechnology Research, Lilly Research Laboratories, Indianapolis, IN 46285, USA.
Kovacevic
S
S
Tinsley
F C
FC
Kussow
C M
CM
Millican
R L
RL
Miller
J R
JR
Hale
J E
JE
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D002942:Circular Dichroism; D003001:Cloning, Molecular; D006801:Humans; D020738:Leptin; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010449:Peptide Mapping; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D018411:Spodoptera; D014357:Trypsin / Q000737:chemistry
D020738:Leptin; D009838:Oligodeoxyribonucleotides; D011506:Proteins; D014357:Trypsin