alexa Efficient secretion of biologically active recombinant OB protein (leptin) in Escherichia coli, purification from the periplasm and characterization.
Bioinformatics & Systems Biology

Bioinformatics & Systems Biology

Current Synthetic and Systems Biology

Author(s): Guisez Y, Fach I, Campfield LA, Smith FJ, Farid A,

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Abstract 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. This article was published in Protein Expr Purif and referenced in Current Synthetic and Systems Biology

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