PDF(Pubmed)
Abstract：
This work unfolds functionalized ABSs composed of FILs ([C2C1Im][C4F9SO3] and [N1112(OH)][C4F9SO3]), mere fluoro-containing ILs ([C2C1Im][CF3SO3] and [C4C1Im][CF3SO3]), known globular protein stabilizers (sucrose and [N1112(OH)][C4F9SO3]), low-molecular-weight carbohydrate (glucose), and even high-charge density salt (K3PO4). The ternary phase diagrams were determined, stressing that FILs highly increased the ability for ABS formation. The functionalized ABSs (FILs vs. mere fluoro-containing ILs) were used to extract lysozyme (Lys). The ABSs\' biphasic regions were screened in terms of protein biocompatibility, analyzing the impact of ABS phase-forming components in Lys by UV-VIS spectrophotometry, CD spectroscopy, fluorescence spectroscopy, DSC, and enzyme assay. Lys partition behavior was characterized in terms of extraction efficiency (% EE). The structure, stability, and function of Lys were maintained or improved throughout the extraction step, as evaluated by CD spectroscopy, DSC, enzyme assay, and SDS-PAGE. Overall, FIL-based ABSs are more versatile and amenable to being tuned by the adequate choice of the phase-forming components and selecting the enriched phase. Binding studies between Lys and ABS phase-forming components were attained by MST, demonstrating the strong interaction between Lys and FILs aggregates. Two of the FIL-based ABSs (30 %wt [C2C1Im][C4F9SO3] + 2 %wt K3PO4 and 30 %wt [C2C1Im][C4F9SO3] + 25 %wt sucrose) allowed the simultaneous purification of Lys and BSA in a single ABS extraction step with high yield (extraction efficiency up to 100%) for both proteins. The purity of both recovered proteins was validated by SDS-PAGE analysis. Even with a high-charge density salt, the FIL-based ABSs developed in this work seem more amenable to be tuned. Lys and BSA were purified through selective partition to opposite phases in a single FIL-based ABS extraction step. FIL-based ABSs are proposed as an improved extraction step for proteins, based on their biocompatibility, customizable properties, and selectivity.
摘要：
这项工作展开了由FIL（[C2C1Im][C4F9SO3]和[N1112（OH）][C4F9SO3]）组成的功能化ABS，仅含氟IL（[C2C1Im][CF3SO3]和[C4C1Im][CF3SO3]），已知的球状蛋白质稳定剂（蔗糖和[N1112（OH）][C4F9SO3]），低分子量碳水化合物（葡萄糖），甚至高电荷密度盐（K3PO4）。确定了三元相图，强调FIL大大提高了ABS形成的能力。功能化ABS(FIL与仅含氟IL）用于提取溶菌酶（Lys）。根据蛋白质生物相容性筛选ABSs双相区域，通过紫外-可见分光光度法分析Lys中ABS相形成成分的影响，CD光谱学,荧光光谱法，DSC,和酶测定。Lys分配行为以提取效率（％EE）为特征。结构,稳定性,在整个提取步骤中保持或改善了Lys的功能，通过CD光谱学评估，DSC,酶测定，和SDS-PAGE。总的来说,基于FIL的ABS更通用并且适合于通过适当选择相形成组分和选择富集相来调节。通过MST获得了Lys和ABS相形成组分之间的结合研究，证明了Lys和FIL聚集体之间的强相互作用。两个基于FIL的ABS（30％wt[C2C1Im][C4F9SO3]2％wtK3PO4和30％wt[C2C1Im][C4F9SO3]25％wt蔗糖）允许同时纯化Lys和BSA在单个ABS提取步骤中，两种蛋白质的收率都很高（提取效率高达100％）。通过SDS-PAGE分析验证两种回收的蛋白质的纯度。即使使用高电荷密度盐，在这项工作中开发的基于FIL的ABS似乎更适合调整。通过在单个基于FIL的ABS提取步骤中选择性分配到相反相来纯化Lys和BSA。基于FIL的ABS被提议作为蛋白质的改进提取步骤，基于它们的生物相容性，可自定义的属性，和选择性。
