Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.
Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China; Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, China.
J Chromatogr A. 2014 May 30;1344:59-65. doi: 10.1016/j.chroma.2014.04.006. Epub 2014 Apr 13.
This work proposed an integrative method of protein refolding and purification by like-charged resin facilitated refolding and metal-chelate affinity adsorption. Hexahistidine-tagged enhanced green fluorescence protein (EGFP) was overexpressed in Escherichia coli as inclusion bodies (IBs), and then the protein was refolded and purified from urea-solubilized IBs by this method. A metal-chelating resin was fabricated by coupling iminodiacetic acid (IDA) to agarose gel (Sepharose FF). The anionic resin was used to facilitate the refolding of like-charged EGFP from IBs. After refolding, nickel ions were introduced for the affinity purification of the target protein by metal-chelating adsorption. It was found that the resin was effective in facilitating EGFP refolding. For 0.1mg/mL EGFP IBs refolding, the fluorescence recovery (FR) by direct dilution was only 64%; addition of only 0.05 g/mL resin increased the FR to over 90%. Moreover, the FR increased with increasing resin concentration. Owning to the shielding effect of the oppositely charged impurities embedded in IBs on the surface charges of the IDA resin, more resin particles were required to exert an aggregation inhibition effect in the IBs protein refolding. Additionally, compared with direct-dilution refolding, inclusion of like-charged resins not only offered an enhanced FR of EGFP, but also bound some opposite-charged contaminant proteins, leading to a preliminary purification effect. Afterwards, the refolded EGFP was recovered by metal-chelating adsorption at an FR of 85% and purity of 93%. This work has thus extended the like-charge facilitated protein refolding strategy to the integrative protein refolding and purification.
这项工作提出了一种通过等电点树脂辅助复性和金属螯合亲和吸附整合蛋白质复性和纯化的方法。六组氨酸标记的增强型绿色荧光蛋白（EGFP）在大肠杆菌中作为包涵体（IBs）过表达，然后通过该方法从尿素溶解的 IBs 中复性和纯化蛋白质。通过将亚氨基二乙酸（IDA）偶联到琼脂糖凝胶（Sepharose FF）上来制备金属螯合树脂。阴离子树脂用于促进带相同电荷的 EGFP 从 IBs 中复性。复性后，引入镍离子通过金属螯合吸附对靶蛋白进行亲和纯化。结果表明，该树脂在促进 EGFP 复性方面非常有效。对于 0.1mg/mL 的 EGFP IBs 复性，直接稀释的荧光恢复（FR）仅为 64％；添加仅 0.05 g/mL 的树脂将 FR 提高到 90％以上。此外，FR 随树脂浓度的增加而增加。由于嵌入 IBs 中的带相反电荷的杂质的屏蔽效应，在 IBs 蛋白复性中需要更多的树脂颗粒来发挥聚集抑制作用。此外，与直接稀释复性相比，包含等电点树脂不仅可以提高 EGFP 的 FR，还可以结合一些带相反电荷的污染物蛋白，从而产生初步的纯化效果。随后，通过金属螯合吸附以 FR 为 85％和纯度为 93％回收复性的 EGFP。因此，这项工作将等电点辅助蛋白复性策略扩展到了整合的蛋白复性和纯化。
