E. coli expression and purification of human and cynomolgus IL-15
E. coli expression and purification of human and cynomolgus IL-15
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DOI：
10.1016/j.pep.2009.05.004
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发表时间：
2009-11-01
影响因子：
1.6
通讯作者：
Mallinder, Philip R.
中科院分区：
文献类型：
--
作者：
Ward, Alison;Anderson, Malcolm;Mallinder, Philip R.
白细胞介素-15 （IL-15）的生理活性表明它可能是一种有用的免疫调节剂，可以激活先天免疫系统，然而，重组成熟IL-15的表达和纯化产量通常很低。在本报告中，优化了一种方法来产生毫克量的这种细胞因子。带n端（His）(6)标签的人IL-15在大肠杆菌中以不溶性蛋白的形式表达。IL-15材料在6 M胍HCl中溶解，然后在含有8 M尿素的缓冲液中进行Ni-NTA层析，从其他细胞蛋白中纯化。使用多组分筛选确定了折叠（His）(6)标记的人IL-15的最佳条件，并将该方法扩大到以其天然构象生产毫克数量的折叠材料，其中两种分子内二硫化物由电喷雾质谱测定。成熟的IL-15通过重组肠激酶裂解生成，随后通过Ni-NTA层析去除。用相同的方法生产成熟的食蟹猴（Macaca fascicularis） IL-15，数量相似。人和食蟹IL-15在两种IL-15依赖性检测中均有活性；小鼠CTLL2细胞增殖和人食蟹细胞CD69上调对全血CD3（-）和CD8（+）淋巴细胞的影响。尽管在氨基酸水平上96%相同，但在两种测定中，人IL-15的效力是食蟹IL-15的10倍。本文描述的方法可用于生产足够数量的成熟IL-15蛋白，用于体内和体外研究，包括x射线晶体学。(C) 2009爱思唯尔公司版权所有。
The physiological activities of Interleukin-15 (IL-15) suggest that it could be useful as an immunomodulator to activate the innate immune system, however, the expression and purification yields of recombinant mature IL-15 have typically been low. In this report, a method was optimised to generate milligram quantities of this cytokine. Human IL-15 with an N-terminal (His)(6)-tag was expressed in Escherichia coli as an insoluble protein. The IL-15 material was purified from other cellular proteins by dissolution in 6 M guanidine HCl, followed by Ni-NTA chromatography in a buffer containing 8 M urea. Use of a multi-component screen identified the optimal conditions for folding (His)(6)-tagged human IL-15 and the method was scaled up to produce milligram quantities of folded material in its native conformation, with two intra-molecular disulphides as determined by electrospray mass spectrometry. Mature IL-15 was generated by cleavage with recombinant enterokinase, which was subsequently removed by Ni-NTA chromatography. Identical methods were used to produce mature cynomolgus monkey (Macaca fascicularis) IL-15 in similar quantities. Human and cynomolgus IL-15 were both active in two IL-15 dependent assays; mouse CTLL2 cell proliferation and human and cynomolgus CD69 upregulation on CD3(-) CD8(+) lymphocytes in whole blood. Despite being 96% identical at the amino acid level the human IL-15 was 10-fold more potent than the cynomolgus IL-15 in both assays. The methods described here are useful for producing both mature IL-15 proteins in sufficient quantity for in vivo and in vitro studies, including X-ray crystallography. (C) 2009 Elsevier Inc. All rights reserved.