title: LPS-protein aggregation influences protein partitioning in aqueous two-phase micellar systems.
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 (%REM(LPS) > 98 %) while GFPuv partitioned preferentially to the micelle-poor phase (K GFP(uv) < 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.
authors: 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. andreml@usp.br
authors: André Moreni
authors: AM
authors: Lopes
authors: Valéria de Carvalho
authors: Vde C
authors: Santos-Ebinuma
authors: Leticia Celia de Lencastre
authors: LC
authors: Novaes
authors: João Vitor Dutra
authors: JV
authors: Molino
authors: Leandro Ramos Souza
authors: LR
authors: Barbosa
authors: Adalberto
authors: A
authors: Pessoa
authors: Carlota de Oliveira
authors: Cde O
authors: Rangel-Yagui
mesh_terms: D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D007700:Kinetics; D008070:Lipopolysaccharides / Q000737:chemistry* / Q000378:metabolism; D008823:Micelles; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D008070:Lipopolysaccharides; D008823:Micelles; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins