LPS–protein aggregation influences protein partitioning in aqueous two-phase micellar systems
LPS–protein aggregation influences protein partitioning in aqueous two-phase micellar systems
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DOI：
10.1007/s00253-013-4922-x
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发表时间：
2013-05
影响因子：
5
通讯作者：
A. Lopes;V. Santos-Ebinuma;Letícia Celia de Lencastre Novaes;J. Molino;Leandro R. S. Barbosa;A. Pessoa;C. O. Rangel-Yagui
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文献类型：
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作者：
A. Lopes;V. Santos-Ebinuma;Letícia Celia de Lencastre Novaes;J. Molino;Leandro R. S. Barbosa;A. Pessoa;C. O. Rangel-Yagui
脂多糖内毒素（LPS）是从革兰氏阴性菌（如大肠杆菌）中纯化的重组肽和蛋白中最常见的致热物质。在这方面，水性两相胶束系统（ATPMS）已经被证明是一个很好的策略，以纯化重组蛋白的药物利益和去除高LPS浓度。在本文中，我们回顾了我们最近在Triton X-114 ATPMS中的蛋白质分配的实验工作，以及一些新的结果，并表明LPS-蛋白质聚集可以影响蛋白质和LPS分配。采用绿色荧光蛋白（GFPuv）作为模型蛋白。ATPMS技术被证明对于高负荷的LPS去除到富胶束相中（%REMLPS> 98%）是有效的，而GFPuv由于排除体积相互作用而优先分配到贫胶束相中（KGFPuv <1.00）。然而，理论预测的蛋白质分配系数值进行了比较，实验获得的，只有在没有LPS的情况下，发现良好的协议。动态光散射测量表明，蛋白质-LPS相互作用的发生，并影响分配过程。我们认为，这种现象应考虑在LPS去除采用任何类型的水两相系统。尽管如此，ATPMS仍然可以被认为是高负荷LPS去除的有效策略，但要知道，由于蛋白质-LPS聚集的存在，可用的排除体积分配理论可能高估分配系数值。
Lipopolysaccharide endotoxins (LPS) are the most common pyrogenic substances in recombinant peptides and proteins purified from Gram-negative bacteria, such asEscherichia 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 (%REMLPS> 98 %) while GFPuv partitioned preferentially to the micelle-poor phase (KGFPuv< 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.