Aqueous two-phase systems containing urea: influence on phase separation and stabilization of protein conformation by phase components.
Aqueous two-phase systems containing urea: influence on phase separation and stabilization of protein conformation by phase components.
复制标题
含有尿素的水两相系统：相组分对相分离和蛋白质构象稳定的影响。
DOI：
10.1021/bp990030
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发表时间：
1999
影响因子：
2.9
通讯作者：
M. Kula
中科院分区：
文献类型：
--
作者：
C. Rämsch;L. B. Kleinelanghorst;E. Knieps;J. Thömmes;M. Kula
在高表达量的重组大肠杆菌发酵过程中，常常形成包涵体。在尿素存在下，已将含水两相系统用于初始回收步骤。为了研究这类体系的相行为，我们在不同尿素浓度和不同PEG分子量下测定了聚乙二醇（PEG）/硫酸钠/尿素/水和PEG/葡聚糖T-500（DEX）/尿素/磷酸盐缓冲液/水的相图。PEG/Na 2SO 4水两相系统可以获得包括高达30%w/w尿素在25 ℃和PEG/葡聚糖T-500高达35%w/w尿素。随着尿素浓度的增加，双峰向更高的浓度位移。尿素的分配系数接近1。以T4-溶菌酶的一个不稳定突变体（V149 T）为研究对象，分析了相组分对酶构象的影响。我们发现，色氨酸的分配是不依赖于尿素的浓度在相系统。
During recombinant Escherichia coli fermentation with high expression levels, inclusion bodies are often formed. Aqueous two-phase systems have been used in the presence of urea for the initial recovery steps. To investigate phase behavior of such systems we determined phase diagrams of poly(ethylene glycol) (PEG)/sodium sulfate/urea/water and PEG/dextran T-500 (DEX)/urea/phosphate buffer/water at different concentrations of urea and different molecular weight of PEG. PEG/Na2SO4 aqueous two-phase systems could be obtained including up to 30% w/w urea at 25 degrees C and PEG/dextran T-500 up to 35% w/w urea. The binodial was displaced toward higher concentrations with increasing urea concentrations. The partition coefficient of urea was near unity. An unstable mutant of T4-lysozyme with an amino acid replacement in the core (V149T) was used to analyze the effect of phase components on the conformation of the enzyme. We showed that partitioning of tryptophan was not dependent on the concentration of urea in the phase system.