Prokaryotic soluble expression and purification of bioactive human fibroblast growth factor 21 using maltose-binding protein.
Prokaryotic soluble expression and purification of bioactive human fibroblast growth factor 21 using maltose-binding protein.
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DOI：
10.1038/s41598-017-16167-x
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发表时间：
2017-11-23
影响因子：
4.6
通讯作者：
Choe H
中科院分区：
文献类型：
--
作者：
Nguyen AN;Song JA;Nguyen MT;Do BH;Kwon GG;Park SS;Yoo J;Jang J;Jin J;Osborn MJ;Jang YJ;Thi Vu TT;Oh HB;Choe H
人成纤维细胞生长因子21(HFGF21)是糖脂代谢动态平衡的重要调节因子。为了在大肠杆菌中高效表达人成纤维细胞生长因子21，通过将hFGF21与以下八个标签之一融合，对其表达和溶解性进行了测试和优化：六组氨酸(His6)、硫氧还蛋白(Trx)、小泛素相关修饰物(SUMO)、谷胱甘肽S转移酶(Gst)、麦芽糖结合蛋白(MBP)、N-利用物质蛋白A(NusA)、人蛋白二硫化物异构酶(PDI)和PDI的b‘a’结构域(PDIB‘A’)。当表达温度为18℃时，每个标签都增加了蛋白质的溶解度，与许多其他被测试的标签不同，MBP显著提高了蛋白质在37℃培养条件下的溶解度。因此，MBP-hFGF21的构建被进一步用于亲和层析纯化的优化。去除标签后，从500mLhFGF21的起始培养中得到纯度为8.1 mg的hFGF21。然后，用质谱学和体外功能分析对该蛋白进行了表征，该蛋白使用了β-klotho报告基因转染的NIH-3T3细胞。这些特性与商品化的hFGF21相似。因此，MBP标签可用于高效原核生产和纯化具有生物活性的hFGF21。
Human fibroblast growth factor 21 (hFGF21) has been characterized as an important regulator of glucose and lipid metabolism homeostasis. Here, to produce hFGF21 efficiently in Escherichia coli, the expression and solubility of hFGF21 were tested and optimised by fusing the protein with one of eight tags: hexahistidine (His6), thioredoxin (Trx), small ubiquitin-related modifier (Sumo), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilisation substance protein A (NusA), human protein disulphide isomerase (PDI), and the b′a′ domain of PDI (PDIb′a′). Each tag increased solubility of the protein when the expression temperature was 18°C. Unlike many other tags that were tested, MBP significantly enhanced the solubility of the protein also in the culture condition at 37°C. Thus, the MBP-hFGF21 construct was further pursued for optimisation of affinity chromatography purification. After tag removal, 8.1 mg of pure hFGF21 was obtained as a final product from 500 mL of starting culture. The protein was then characterised by mass spectroscopy and an in vitro functional assay using NIH-3T3 cells transfected with a β-klotho reporter gene. These characteristics are similar to those of commercial hFGF21. Thus, the MBP tag is useful for efficient prokaryotic production and purification of bioactive hFGF21.
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DOI：
10.1073/pnas.0701600104
发表时间：
2007-05-01
影响因子：
11.1
作者：
Ogawa, Yasushi;Kurosu, Hiroshi;Kuro-o, Makoto
通讯作者：
Kuro-o, Makoto
麦芽糖结合蛋白和蛋白质二硫化物异构酶对生物活性人粒细胞刺激因子的生物活性人粒细胞群刺激因子的纯化。
影响因子：
3.7
作者：
Do BH;Ryu HB;Hoang P;Koo BK;Choe H
通讯作者：
Choe H
影响因子：
5.6
作者：
Micanovic, Radmila;Raches, David W.;Kharitonenkov, Alexei
通讯作者：
Kharitonenkov, Alexei
影响因子：
5.6
作者：
Kharitonenkov, Alexei;Dunbar, James D.;Shanafelt, Armen B.
通讯作者：
Shanafelt, Armen B.
脂质A结构修饰内毒素活性降低的大肠杆菌突变体的构建
影响因子：
5.4
作者：
Liu Q;Li Y;Zhao X;Yang X;Liu Q;Kong Q
通讯作者：
Kong Q