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首页> 外文期刊>Applied Microbiology and Biotechnology >LPS-protein aggregation influences protein partitioning in aqueous two-phase micellar systems
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LPS-protein aggregation influences protein partitioning in aqueous two-phase micellar systems

机译：LPS蛋白质聚集影响水性两相胶束系统中的蛋白质分配

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摘要

Lipopolysaccharide endotoxins (LPS) are the most common pyrogenic substances in recombinant peptides and proteins purified from Gram-negative bacteria, such as Escherichia coli. In this respect, aqueous two-phase micellar systems (ATPMS) have already proven to be a good strategy to purify recombinant proteins of pharmaceutical interest and remove high LPS concentrations. In this paper, we review our recent experimental work in protein partitioning in Triton X-114 ATPMS altogether with some new results and show that LPS-protein aggregation can influence both protein and LPS partitioning. Green fluorescent protein (GFPuv) was employed as a model protein. The ATPMS technology proved to be effective for high loads of LPS removal into the micelle-rich phase (%REM_(LPS) > 98 %) while GFPuv partitioned preferentially to the micelle-poor phase (K _(GFPuv) < 1.00) due to the excluded-volume interactions. However, theoretically predicted protein partition coefficient values were compared with experimentally obtained ones, and good agreement was found only in the absence of LPS. Dynamic light scattering measurements showed that protein-LPS interactions were taking place and influenced the partitioning process. We believe that this phenomenon should be considered in LPS removal employing any kind of aqueous two-phase system. Nonetheless, ATPMS can still be considered as an efficient strategy for high loads of LPS removal, but being aware that the excluded-volume partitioning theory available might overestimate partition coefficient values due to the presence of protein-LPS aggregation.
机译：脂多糖内毒素（LPS）是从革兰氏阴性细菌（例如大肠杆菌）纯化的重组肽和蛋白质中最常见的热原物质。在这方面，已经证明含水两相胶束系统（ATPMS）是纯化具有药用价值的重组蛋白并去除高LPS浓度的良好策略。在本文中，我们回顾了我们最近在Triton X-114 ATPMS中进行蛋白质分配的实验工作，并获得了一些新结果，并表明LPS-蛋白质聚集既可以影响蛋白质分配又可以影响LPS分配。绿色荧光蛋白（GFPuv）被用作模型蛋白。事实证明，ATPMS技术可有效去除高负荷的LPS进入富含胶束的相（％REM_（LPS）> 98％），而GFPuv则优先分配至缺乏胶束的相（K _（GFPuv）<1.00），这是由于排除体积的互动。但是，将理论上预测的蛋白质分配系数值与实验获得的值进行了比较，并且只有在没有LPS的情况下才能发现良好的一致性。动态光散射测量表明蛋白质-LPS相互作用正在发生并影响分配过程。我们相信在采用任何一种水相两相系统去除LPS时都应考虑这种现象。尽管如此，ATPMS仍然可以被认为是高负荷LPS去除的有效策略，但是请注意，由于存在蛋白质-LPS聚集，因此可用的排除体积分配理论可能会高估分配系数值。

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