Abstract
The application of recombinant (His)(6)-tagged proteins in cell culture assays is associated with problems due to lipopolysaccharide (LPS) contamination. LPS stimulates cells of the immune system, thereby masking antigen-specific activation of T cells. Due to the affinity of LPS for histidine it is associated with difficulties to remove LPS from recombinant (His)(6)-tagged proteins. Here we describe that the Triton X-114 phase separation method can be used to remove LPS from (His)(6)-tagged proteins and that the recombinant proteins retain their biological activity.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Allergens / chemistry
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Allergens / genetics
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Allergens / metabolism
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Allergens / pharmacology*
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Antigens, Plant
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Basophils / drug effects*
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Basophils / immunology
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Cells, Cultured
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Cloning, Molecular
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Dose-Response Relationship, Drug
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Glycoproteins / chemistry
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Glycoproteins / genetics
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Glycoproteins / metabolism
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Glycoproteins / pharmacology*
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Histamine Release / drug effects
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Histidine / metabolism
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Humans
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Interleukin-10 / metabolism
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Leukocytes, Mononuclear / drug effects*
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Leukocytes, Mononuclear / immunology
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Lipopolysaccharides / isolation & purification*
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Lipopolysaccharides / metabolism
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Lipopolysaccharides / pharmacology
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Membrane Proteins
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Octoxynol
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Plant Proteins / chemistry
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Plant Proteins / genetics
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Plant Proteins / metabolism
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Plant Proteins / pharmacology*
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Polyethylene Glycols / chemistry
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Protein Binding
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Recombinant Proteins / metabolism
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T-Lymphocytes / drug effects*
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T-Lymphocytes / immunology
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Time Factors
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Tumor Necrosis Factor-alpha / metabolism
Substances
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Allergens
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Antigens, Plant
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Ara h 1 protein, Arachis hypogaea
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Glycoproteins
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IL10 protein, human
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Lipopolysaccharides
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Membrane Proteins
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Plant Proteins
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Recombinant Proteins
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Tumor Necrosis Factor-alpha
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Interleukin-10
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Polyethylene Glycols
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Histidine
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Octoxynol
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Nonidet P-40