Xiao Chunlan, Liu Junyi, Tang Yanchun, Chen Junyong, Wu Xiaopeng, Bi Feng, Zhang Jing
Institute of Molecular Medicine and Bio-Pharmaceutical Engineering Research Center, Nanjing University, Nanjing, People's Republic of China.
College of Arts and Sciences, Emory University, Atlanta, GA, USA.
Biotechnol Appl Biochem. 2017 Jan;64(1):43-49. doi: 10.1002/bab.1458. Epub 2016 Mar 10.
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.
Nesfatin-1是一种新发现的饱腹感分子，主要在下丘脑核中表达。它能抑制短期和长期食欲。通过PCR重叠的六个编码nesfatin-1的合成脱氧寡核苷酸，在其六组氨酸标签多克隆位点序列之间插入肠激酶识别位点后，被克隆到pET28a载体中。重组质粒被转化到大肠杆菌Rosetta菌株中以表达融合蛋白，该融合蛋白占总细胞蛋白的27%。经镍琼脂糖亲和层析纯化后，用肠激酶处理融合蛋白以释放nesfatin-1。nesfatin-1样品再用反相高效液相色谱（HPLC）进一步纯化，并用质谱法测定其分子量。重组nesfatin-1的生物活性也使用体内动物模型进行评估。本文所述方法有望从1升大肠杆菌摇瓶培养物中产生约8毫克生物活性nesfatin-1，其纯度超过98%，这可被视为一种简单且经济高效的合成nesfatin-1的方法。
