The biological activity of a recombinantly expressed (His)(6)-tagged peanut allergen (rAra h 1) is unaffected by endotoxin removal.
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.
Jensen
Louise Bjerremann
LB
Department of Agricultural Sciences, Faculty of Life Sciences, University of Copenhagen, Denmark. lbjj@life.ku.dk
Torp
Anna Maria
AM
Andersen
Sven Bode
SB
Skov
Per Stahl
PS
Poulsen
Lars K
LK
Knol
Edward F
EF
van Hoffen
Els
E
D000485:Allergens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D052179:Antigens, Plant; D001491:Basophils / Q000187:drug effects* / Q000276:immunology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D006636:Histamine Release / Q000187:drug effects; D006639:Histidine / Q000378:metabolism; D006801:Humans; D016753:Interleukin-10 / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000187:drug effects* / Q000276:immunology; D008070:Lipopolysaccharides / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D008565:Membrane Proteins; D017830:Octoxynol; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D011092:Polyethylene Glycols / Q000737:chemistry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013601:T-Lymphocytes / Q000187:drug effects* / Q000276:immunology; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D000485:Allergens; D052179:Antigens, Plant; C500889:Ara h 1 protein, Arachis hypogaea; D006023:Glycoproteins; C508609:IL10 protein, human; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D011092:Polyethylene Glycols; D006639:Histidine; D017830:Octoxynol; C010615:Nonidet P-40