Design of functional guanidinium ionic liquid aqueous two-phase systems for the efficient purification of protein
Design of functional guanidinium ionic liquid aqueous two-phase systems for the efficient purification of protein
复制标题
用于高效纯化蛋白质的功能性胍离子液体水两相系统的设计
DOI：
10.1016/j.aca.2014.01.030
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发表时间：
2014-03-07
影响因子：
6.2
通讯作者：
Xu, Kaijia
中科院分区：
文献类型：
--
作者：
Ding, Xueqin;Wang, Yuzhi;Xu, Kaijia
以1，1，3，3-四甲基胍为原料，设计并合成了一系列新型阳离子功能化六烷基胍离子液体和阴离子功能化四烷基胍离子液体。离子液体的结构经核磁共振氢谱（H-1 NMR）和碳谱（13 C NMR）确证，产率均在90%以上。首次使用这些功能性胍离子液体和磷酸盐溶液设计了功能性胍离子液体含水两相系统（FGIL-ATPS）用于蛋白质纯化。相分离后，蛋白质转移到富含IL的相中，并通过使用紫外可见（UV-vis）分光光度计测量278 nm处的吸光度来测定蛋白质的浓度。比较了FGIL-ATPS与普通离子液体双水相体系（IL-ATPS）的优点。该方法已应用于溶菌酶、胰蛋白酶、卵清蛋白和牛血清白蛋白的纯化。通过单因素实验考察了离子液体用量、盐溶液浓度、温度和蛋白质用量对酶解效果的影响。净化效率达到97.05%。利用紫外可见分光光度计、傅里叶变换红外光谱（FT-IR）和圆二色谱（CD）对实验过程中蛋白质的二级结构进行了观察。考察了该方法的精密度、稳定性和重复性。通过动态光散射（DLS）、电导率测定和透射电镜（TEM）研究了其净化机理。结果表明，聚集和拥抱现象在蛋白质的纯化过程中起着重要作用。这些结果表明，FGIL-ATPS在蛋白质纯化方面具有巨大的潜力。(C)2014年爱思唯尔B。V.保留所有权利。
A series of novel cationic functional hexaalkylguanidinium ionic liquids and anionic functional tetraalkylguanidinium ionic liquids have been devised and synthesized based on 1,1,3,3-tetramethylguanidine. The structures of the ionic liquids (ILs) were confirmed by H-1 nuclear magnetic resonance (H-1 NMR) and 13C nuclear magnetic resonance (13C NMR) and the production yields were all above 90%. Functional guanidinium ionic liquid aqueous two-phase systems (FGIL-ATPSs) have been first designed with these functional guanidinium ILs and phosphate solution for the purification of protein. After phase separation, proteins had transferred into the IL-rich phase and the concentrations of proteins were determined by measuring the absorbance at 278 nm using an ultra violet visible (UV-vis) spectrophotometer. The advantages of FGIL-ATPSs were compared with ordinary ionic liquid aqueous two-phase systems (IL-ATPSs). The proposed FGIL-ATPS has been applied to purify lysozyme, trypsin, ovalbumin and bovine serum albumin. Single factor experiments were used to research the effects of the process, such as the amount of ionic liquid (IL), the concentration of salt solution, temperature and the amount of protein. The purification efficiency reaches to 97.05%. The secondary structure of protein during the experimental process was observed upon investigation using UV-vis spectrophotometer, Fourier-transform infrared spectroscopy (FT-IR) and circular dichroism spectrum (CD spectrum). The precision, stability and repeatability of the process were investigated. The mechanisms of purification were researched by dynamic light scattering (DLS), determination of the conductivity and transmission electron microscopy (TEM). It was suggested that aggregation and embrace phenomenon play a significant role in the purification of proteins. All the results show that FGIL-ATPSs have huge potential to offer new possibility in the purification of proteins. (C) 2014 Elsevier B. V. All rights reserved.