Shao Mingcong, Xiu Lili, Zhang Haijiang, Huang Jianying, Gong Xingwen
a College of Food Science and Biotechnology , Zhejiang Gongshang University , Hangzhou , P. R. China.
b Jiangsu Key Laboratory of Regional Resource Exploitation and Medicinal Research , Huaiyin Institute of Technology , Huaian , P. R. China.
Prep Biochem Biotechnol. 2018 Apr 21;48(4):352-360. doi: 10.1080/10826068.2018.1446154. Epub 2018 Apr 10.
Chitosan/cellulose-based beads (CCBs) for the affinity purification of histidine-tagged proteins were prepared from chitosan/cellulose dissolved in ionic liquid as a solvent, and their structures were characterized by Fourier transform infrared spectroscopy, transmission electron microscopy, and thermogravimetric analysis. The affinity purification was used to separate hexahistidine-tagged (his-tagged) enhanced green fluorescent protein (EGFP) from Escherichia coli. The results showed that Zn-CCB exhibited more specific adsorption capacity toward the target protein compared with Ni-CCB and Cu-CCB. The maximum adsorption of EGFP was 1.84 mg/g of Zn-CCB, with 90% purity under the optimized conditions (ionic strength (1.0 M NaCl), pH (7.2) and imidazole concentration (500 mM)). In addition, a regeneration method for the sorbent was further developed by washing with ethylenediaminetetraacetic acid disodium and then reimmobilizing with metal ions. This technique is an alternative method for the purification of his-tagged proteins, making the process more economical, fast, stable, and large batch.
以壳聚糖/纤维素为基础的磁珠（CCBs）用于亲和纯化组氨酸标签蛋白，它是由溶解在离子液体中的壳聚糖/纤维素作为溶剂制备而成，其结构通过傅里叶变换红外光谱、透射电子显微镜和热重分析进行表征。亲和纯化用于从大肠杆菌中分离六组氨酸标签（His标签）的增强型绿色荧光蛋白（EGFP）。结果表明，与镍基CCB和铜基CCB相比，锌基CCB对目标蛋白表现出更高的特异性吸附能力。在优化条件（离子强度（1.0 M NaCl）、pH值（7.2）和咪唑浓度（500 mM））下，EGFP在锌基CCB上的最大吸附量为1.84 mg/g，纯度为90%。此外，通过用乙二胺四乙酸二钠洗涤，然后用金属离子重新固定，进一步开发了一种吸附剂再生方法。该技术是纯化His标签蛋白的一种替代方法，使该过程更经济、快速、稳定且可大规模进行。
