title: 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.
authors.affiliation: Institute of Molecular Medicine and Bio-Pharmaceutical Engineering Research Center, Nanjing University, Nanjing, People's Republic of China.
authors.forename: Chunlan
authors.initials: C
authors.lastname: Xiao
authors.affiliation: College of Arts and Sciences, Emory University, Atlanta, GA, USA.
authors.forename: Junyi
authors.initials: J
authors.lastname: Liu
authors.affiliation: Institute of Molecular Medicine and Bio-Pharmaceutical Engineering Research Center, Nanjing University, Nanjing, People's Republic of China.
authors.forename: Yanchun
authors.initials: Y
authors.lastname: Tang
authors.affiliation: Institute of Molecular Medicine and Bio-Pharmaceutical Engineering Research Center, Nanjing University, Nanjing, People's Republic of China.
authors.forename: Junyong
authors.initials: J
authors.lastname: Chen
authors.affiliation: Institute of Molecular Medicine and Bio-Pharmaceutical Engineering Research Center, Nanjing University, Nanjing, People's Republic of China.
authors.forename: Xiaopeng
authors.initials: X
authors.lastname: Wu
authors.affiliation: Institute of Molecular Medicine and Bio-Pharmaceutical Engineering Research Center, Nanjing University, Nanjing, People's Republic of China.
authors.forename: Feng
authors.initials: F
authors.lastname: Bi
authors.affiliation: Institute of Molecular Medicine and Bio-Pharmaceutical Engineering Research Center, Nanjing University, Nanjing, People's Republic of China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000818:Animals; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D051379:Mice; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D000080866:Nucleobindins; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins; D009419:Nerve Tissue Proteins; C000630651:Nucb1 protein, mouse; D000080866:Nucleobindins; D011994:Recombinant Proteins
keywords: anti-hyperglycemia; nucb2; nucleobindin; recombinant technology; satiety