Utilization of Arg-elution method for FLAG-tag based chromatography.
FLAG-tag is one of the commonly used purification technologies for recombinant proteins. An antibody, M2, specifically binds to the FLAG-tag whether it is attached to N- or C-terminus of proteins to be purified. The bound proteins are generally eluted by competition with a large excess of free FLAG peptide. This requires synthetic FLAG peptide and also removal of bound FLAG peptide for M2 column regeneration. We have shown before that arginine at mild pH can effectively dissociate protein-protein or protein-ligand interactions, e.g. in Protein-A, antigen and dye-affinity chromatography. We have tested here elution of FLAG-fused proteins by arginine for columns of M2-immobilized resin using several proteins in comparison with competitive elution by FLAG peptide or low pH glycine buffer. Active and folded proteins were successfully and effectively eluted using 0.5-1M arginine at pH 3.5-4.4, as reported in this paper.
Futatsumori-Sugai
Mutsumi
M
Department of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa 277-8562, Japan.
Abe
Ryota
R
Watanabe
Masato
M
Kudou
Motonori
M
Yamamoto
Tadashi
T
Ejima
Daisuke
D
Arakawa
Tsutomu
T
Tsumoto
Kouhei
K
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D001120:Arginine / Q000737:chemistry*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012831:Bombyx; D002152:Calorimetry, Differential Scanning; D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel; D006863:Hydrogen-Ion Concentration; D019476:Insect Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D048057:Mitogen-Activated Protein Kinase 10 / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D019476:Insect Proteins; C079726:MsbA protein, Bacteria; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D001120:Arginine; C085706:FLAG peptide; D048057:Mitogen-Activated Protein Kinase 10