Recombinant expression, isotope labeling, refolding, and purification of an antimicrobial peptide, piscidin
Recombinant expression, isotope labeling, refolding, and purification of an antimicrobial peptide, piscidin
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DOI：
10.1016/j.pep.2006.07.010
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发表时间：
2007-02-01
影响因子：
1.6
通讯作者：
Chae, Young Kee
中科院分区：
文献类型：
--
作者：
Moon, Won Jae;Hwang, Dong Kyu;Chae, Young Kee
抗菌肽 piscidin 在大肠杆菌中以与泛素分子融合的形式过表达，并使用固定化金属亲和层析 (IMAC) 纯化融合蛋白。使用酵母泛素水解酶 (YUH) 将肽从其融合伴侣中释放出来，随后通过反相色谱进行纯化。鱼素的表达和纯化过程遇到了几个问题，例如诱导肽产生时细菌细胞的裂解、细菌细胞内融合蛋白的不必要的裂解以及在水性环境中高度聚集的倾向。通过使用泛素作为 piscidin 的融合伴侣、在较低温度下培养细胞以及改变纯化步骤的顺序，这些问题得到了缓解。融合蛋白和肽的产量分别约为每升 LB 或基本培养基 15 毫克和 1.5 毫克。 piscidin 的重组表达和纯化将使其能够使用多维核磁共振波谱进行结构和动态研究。 (c) 2006 Elsevier Inc. 保留所有权利。
An antimicrobial peptide, piscidin, was overexpressed as a fused form with the ubiquitin molecule in Escherichia coli, and the fusion protein was purified using immobilized metal affinity chromatography (IMAC). The peptide was released from its fusion partner by using yeast ubiquitin hydrolase (YUH), and subsequently purified by reverse phase chromatography. The expression and purification process of piscidin encountered several problems such as the lysis of the bacterial cell upon induction of the peptide production, the unwanted cleavage of the fusion protein inside the bacterial cell, and high tendency to aggregate in the aqueous environment. Such problems were alleviated by employing ubiquitin as a fusion partner for piscidin, growing the cells at a lower temperature, and changing the order of the purification steps. The yields of the fusion protein and the peptide were around 15 and 1.5 mg per liter of LB or minimal medium, respectively. The recombinant expression and purification of piscidin will enable its structural and dynamic studies using multidimensional NMR spectroscopy. (c) 2006 Elsevier Inc. All rights reserved.