We found a match
Your institution may have access to this item. Find your institution then sign in to continue.
- Title
Expression, purification, and characterization of mouse nesfatin-1 in Escherichia coli.
- Authors
Xiao, Chunlan; Liu, Junyi; Tang, Yanchun; Chen, Junyong; Wu, Xiaopeng; Bi, Feng; Zhang, Jing
- 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.
- Subjects
THERMOPHILIC microorganisms; ESCHERICHIA coli DNA; ESCHERICHIA coli transmission; HYPERGLYCEMIA prevention; HYPERGLYCEMIA treatment; RECOMBINANT antibodies
- Publication
Biotechnology & Applied Biochemistry, 2017, Vol 64, Issue 1, p43
- ISSN
0885-4513
- Publication type
Article
- DOI
10.1002/bab.1458