Expression, purification, and characterization of mouse nesfatin‐1 in Escherichia coli

Abstract 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 were 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 Escherichia 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 high performance liquid chromatography (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 1‐L shaking flask culture of E. coli , which can be considered as an easy and cost‐effective way to synthesize nesfatin‐1.