Production of leptin in Escherichia coli: a comparison of methods.
A procedure is described for gram-scale refolding of Escherichia coli-derived human leptin inclusion bodies. Refolding was achieved by gradually reducing denaturant using a diafiltration method. Refolded leptin is characterized by in vivo modulation of food intake, reduction in body weight, and lowering of insulin and glucose levels in ob/ob mice. In addition, refolded leptin is characterized by radioimmunoassay (RIA) and activation of the leptin receptor in a cell-based assay. For comparison we also refolded leptin by a simple dilution method and produced periplasmic derived leptin, which did not require ex vivo folding. Leptin produced by these three methods and leptin obtained from commercial sources were compared using the RIA and the cell-based assay and appeared to be of comparable quality and potency.
Varnerin
J P
JP
Department of Obesity Research, Merck Research Laboratories, Rahway, New Jersey, 07065, USA.
Smith
T
T
Rosenblum
C I
CI
Vongs
A
A
Murphy
B A
BA
Nunes
C
C
Mellin
T N
TN
King
J J
JJ
Burgess
B W
BW
Junker
B
B
Chou
M
M
Hey
P
P
Frazier
E
E
MacIntyre
D E
DE
Van der Ploeg
L H
LH
Tota
M R
MR
D000818:Animals; D001681:Biological Assay; D002460:Cell Line; D004731:Endotoxins / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D005374:Filtration; D005822:Genetic Vectors / Q000235:genetics; D006639:Histidine; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D020738:Leptin; D051379:Mice; D008817:Mice, Mutant Strains; D008820:Mice, Obese; D010455:Peptides / Q000737:chemistry; D014176:Protein Biosynthesis; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D011863:Radioimmunoassay; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D015431:Weight Loss / Q000187:drug effects
D004731:Endotoxins; D020738:Leptin; D010455:Peptides; D011506:Proteins; D011993:Recombinant Fusion Proteins; C033223:polyhistidine; D006639:Histidine