Expression, purification, and characterization of recombinant human H-chain ferritin

Wenyan Zou, Xiaoyu Liu, Dian-hua Chen, Jie Wang, Xi Zhao, Jiahuang Li, Lina Ji, Z. Hua
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引用次数: 5

Abstract

ABSTRACT Based on their nanocage architectures, ferritins show their potential applications in medical imaging and therapeutic delivery systems. However, the recombinant human H-chain ferritin (rHF) is prone to form inclusion bodies in Escherichia coli. In our study, the cDNA of rHF was cloned into plasmid pET28a under the control of a T7 promoter. Molecular chaperones, including GroES, GroEL, and trigger factor, were coexpressed with rHF to facilitate its correct folding. The results showed that the solubility of rHF was increased more than threefold with the help of molecular chaperones. Taking advantages of its N-terminal His-tag, rHF was then purified with Ni-affinity chromatography. With a yield of 15 mg/L from bacterial culture, the purified rHF was analyzed by circular dichroism spectrometry for its secondary structure. Moreover, the rHF nanocages were characterized by transmission electron microscopy and dynamic light scattering. Our results indicate that rHF is able to self-assemble into nanocages with a narrow size distribution.
重组人h链铁蛋白的表达、纯化和表征
基于其纳米笼结构,铁蛋白在医学成像和治疗递送系统中显示出其潜在的应用。然而,重组人h链铁蛋白(rHF)在大肠杆菌中容易形成包涵体。本研究在T7启动子的控制下,将rHF cDNA克隆到质粒pET28a中。分子伴侣包括GroES, GroEL和触发因子与rHF共表达,以促进其正确折叠。结果表明,在分子伴侣的帮助下,rHF的溶解度提高了三倍以上。利用其n端his标签,用ni亲和层析纯化rHF。细菌培养产量为15 mg/L,用圆二色光谱法分析纯化的rHF的二级结构。利用透射电镜和动态光散射对所制备的rHF纳米笼进行了表征。我们的研究结果表明,rHF能够自组装成具有窄尺寸分布的纳米笼。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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