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.

Study of the effects of nesfatin-1 on gastric function in obese rats

“…Nesfatin-1, an anorectic peptide first discovered in 2006, has been proven as an inhibitor of food intake[5-8]. Nesfatin-1 can be expressed in peripheral organs, such as the gastrointestinal tract, and plays a role in the regulation of many physiological processes, such as carbohydrate metabolism, immunity, and the digestive tract[9-13]. Studies have shown that nesfatin-1 mRNA, together with ghrelin somatostatin and histidine decarboxylase, regulate gastric acid secretion and are co-expressed in oxyntic glands in the fundus of the stomach; this means that nesfatin-1 may be also involved in gastric acid secretion[14-16].…”