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LPS–protein aggregation influences protein partitioning in aqueous two-phase micellar systems

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Abstract

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 (%REMLPS > 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.

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Acknowledgments

This research was supported by grants from the Coordination for Higher Level Graduate Improvements (CAPES/Brazil), National Council for Scientific and Technological Development (CNPq/Brazil), and State of São Paulo Research Foundation (FAPESP/Brazil).

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Authors and Affiliations

  1. Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of São Paulo-FCF/USP, Av. Prof. Lineu Prestes, 580, B16 Cidade Universitária, 05508-000, São Paulo, SP, Brazil

    André Moreni Lopes, Valéria de Carvalho Santos-Ebinuma, Leticia Celia de Lencastre Novaes, João Vitor Dutra Molino & Adalberto Pessoa Jr

  2. Department of General Physics, Institute of Physics-University of São Paulo, São Paulo, Brazil

    Leandro Ramos Souza Barbosa

  3. Department of Pharmacy, School of Pharmaceutical Sciences, University of São Paulo-FCF/USP, São Paulo, Brazil

    Carlota de Oliveira Rangel-Yagui

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  1. André Moreni Lopes
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  2. Valéria de Carvalho Santos-Ebinuma
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  3. Leticia Celia de Lencastre Novaes
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  4. João Vitor Dutra Molino
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  6. Adalberto Pessoa Jr
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  7. Carlota de Oliveira Rangel-Yagui
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Correspondence to André Moreni Lopes.

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Lopes, A.M., Santos-Ebinuma, V.d.C., Novaes, L.C.d.L. et al. LPS–protein aggregation influences protein partitioning in aqueous two-phase micellar systems. Appl Microbiol Biotechnol 97, 6201–6209 (2013). https://doi.org/10.1007/s00253-013-4922-x

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  • DOI: https://doi.org/10.1007/s00253-013-4922-x

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