Nicotinamide phosphoribosyltransferase purification using SUMO expression system.
Nicotinamide phosphoribosyltransferase purification using SUMO expression system.
复制标题
DOI：
10.1016/j.ab.2020.113597
复制
发表时间：
2020-06-01
影响因子：
2.9
通讯作者：
Garcia JGN
中科院分区：
文献类型：
--
作者：
Molugu TR;Oita RC;Chawla U;Camp SM;Brown MF;Garcia JGN
烟酰胺磷酸核糖转移酶(NAMPT)是烟酰胺腺嘌呤二核苷酸合成所需的挽救途径中的限速酶。分泌的NAMPT蛋白作为一种主要的调节细胞因子参与了进化保守的炎症网络的激活。对NAMPT作为损伤相关分子模式蛋白(DAMP)作用的认识，已将其活性与Toll样受体4(TLR4)结合和炎症级联激活等多种疾病联系起来。由于用于体外和体内研究的纯化蛋白的可用性有限，目前缺乏关于NAMPT蛋白功能的确切模式的信息。在这里，我们报告了使用pET-SUMO表达载体在含有用于纯化的六组氨酸标签的大肠杆菌菌株Shuffle中成功地表达NAMPT。用Ulp1酶切SUMO和His标签，用固定化金属亲和层析纯化蛋白。蛋白质产率为~4 mg/L，圆二色谱初步表征了蛋白质的二级结构元素，而动态光散射表明蛋白质中存在低聚单元。NAMPT-SUMO显示了一种主要的二聚体蛋白，具有功能性酶活性。最后，我们报道了N-十二烷基-β-D-麦芽吡喃糖苷(DDM)洗涤剂中的单体增溶作用，从而增加了进一步研究结构和功能的机会。
Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme in the salvage pathway required for nicotinamide adenine dinucleotide synthesis. The secreted NAMPT protein serves as a master regulatory cytokine involved in activation of evolutionarily-conserved inflammatory networks. Appreciation of the role of NAMPT as a damage-associated molecular pattern protein (DAMP) has linked its activities to several disorders via toll-like receptor 4 (TLR4) binding and inflammatory cascade activation. Information is currently lacking concerning the precise mode of the NAMPT protein functionality due to limited availability of purified protein for use in in vitro and in vivo studies. Here we report successful NAMPT expression using the pET-SUMO expression vector in E. coli strain SHuffle containing a hexa-His tag for purification. The Ulp1 protease was used to cleave the SUMO and hexa-His tags, and the protein was purified by immobilized-metal affinity chromatography. The protein yield was ~4 mg/L and initial biophysical characterization of the protein using circular dichroism revealed the secondary structural elements, while dynamic light scattering demonstrated the presence of oligomeric units. The NAMPT-SUMO showed a predominantly dimeric protein with functional enzymatic activity. Finally, we report NAMPT solubilization in n-dodecyl-β-D-maltopyranoside (DDM) detergent in monomeric form, thus enhancing the opportunity for further structural and functional investigations.
登录
查看更多内容
DOI：
10.1016/j.biocel.2012.11.002
发表时间：
2013-02-01
影响因子：
4
作者：
Chen, Xin-Yu;Zhang, Hong-Sheng;Ruan, Zheng
通讯作者：
Ruan, Zheng
影响因子：
2.7
作者：
Liu, Zixuan;Zhang, Jizhou;Xiao, Yechen
通讯作者：
Xiao, Yechen
DOI：
10.1002/anie.201508648
发表时间：
2016-01-11
期刊：
Angewandte Chemie (International ed. in English)
影响因子：
--
作者：
Chawla U;Jiang Y;Zheng W;Kuang L;Perera SM;Pitman MC;Brown MF;Liang H
通讯作者：
Liang H
DOI：
10.1007/978-3-642-17214-4_7
发表时间：
2011-01-01
期刊：
DIABETES - PERSPECTIVES IN DRUG THERAPY
影响因子：
--
作者：
Garten, Antje;Petzold, Stefanie;Kiess, Wieland
通讯作者：
Kiess, Wieland
通过与 SUMO 融合表达和纯化功能性 HMGB1 A 盒。
影响因子：
3.4
作者：
Ge WS;Fan JG;Chen YW;Xu LM
通讯作者：
Xu LM