Expression, purification and characterization of mouse nesfatin-1 in E. coli.

Nesfatin-1 is a newly discovered satiety molecule expressed mainly in the hypothalamic nuclei. It suppresses both short term and long term appetite. Six synthetic deoxyoligonucleotides overlapped by PCR encoding nesfatin-1 was cloned into a pET28a vector after the hexa-histidine tagged Multiple Cloning Sites sequence with an enterokinase recognition site incorporated in-between. The recombinant plasmid was transformed into E. coli strain Rosetta to express the fusion protein, which constituted 27% of the total cell proteins. After purified by Ni-Sepharose affinity chromatography, the fusion protein was treated with enterokinase to release nesfatin-1. The nesfatin-1 sample was further purified with reverse phase HPLC, and its molecular weight was determined by mass spectrometry. The biological activities of recombinant nesfatin-1 were also assessed using in vivo animal models. The method described here promises to produce about 8 mg biologically active nesfatin-1 with homogeneity over 98% from one liter shaking flask culture of E. coli, which can be considered as an easy and cost-effective way to synthesize nesfatin-1. This article is protected by copyright. All rights reserved.