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). T he hybrid genes were preceded by a ribosome binding site (RES) and wer e expressed under transcriptional control of both the lipoprotein prom oter (P-1pp) and the lac promoter-operator (POlac). The recombinant fu sion proteins were efficiently expressed and exported into the peripla smic compartment of E. coli cells hom where they were recovered by osm otic shock as soluble mature polypeptides with the sOMP A precisely re moved. Recombinant mOB and hOB proteins were also produced in Sf 9 ins ect cells using the baculovirus expression system. Milligram quantitie s of both proteins were purified to homogeneity using ion-exchange, hy drophobic interaction chromatography and gel filtration and were found to be biologically active and to have antiobesity effects upon testin g in genetically obese ob/ob mice. (C) 1998 Academic Press.