Efficient secretion of biologically active recombinant OB protein (leptin) in Escherichia coli, purification from the periplasm and characterization.
The genes encoding the mature forms of mouse (mOB) and human OB (hOB) protein (also called leptin) were fused to the secretion signal coding sequence of the Escherichia coli outer membrane protein A (sOMP A). The hybrid genes were preceded by a ribosome binding site (RBS) and were expressed under transcriptional control of both the lipoprotein promoter (Plpp) and the lac promoter-operator (POlac). The recombinant fusion proteins were efficiently expressed and exported into the periplasmic compartment of E. coli cells from where they were recovered by osmotic shock as soluble mature polypeptides with the sOMP A precisely removed. Recombinant mOB and hOB proteins were also produced in Sf9 insect cells using the baculovirus expression system. Milligram quantities of both proteins were purified to homogeneity using ion-exchange, hydrophobic interaction chromatography and gel filtration and were found to be biologically active and to have antiobesity effects upon testing in genetically obese ob/ob mice.
Guisez
Y
Y
Roche Research Gent, Belgium.
Faché
I
I
Campfield
L A
LA
Smith
F J
FJ
Farid
A
A
Plaetinck
G
G
Van der Heyden
J
J
Tavernier
J
J
Fiers
W
W
Burn
P
P
Devos
R
R
D000818:Animals; D001835:Body Weight / Q000187:drug effects* / Q000502:physiology; D002460:Cell Line; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D004195:Disease Models, Animal; D004435:Eating / Q000187:drug effects* / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D006801:Humans; D007274:Injections, Intraperitoneal; D007276:Injections, Intraventricular; D020738:Leptin; D051379:Mice; D008820:Mice, Obese; D009765:Obesity / Q000188:drug therapy / Q000503:physiopathology; D019897:Periplasm; D011506:Proteins / Q000008:administration & dosage / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D018411:Spodoptera
D020738:Leptin; D011506:Proteins; D011994:Recombinant Proteins