A heme fusion tag for protein affinity purification and quantification.
We report a novel affinity-based purification method for proteins expressed in Escherichia coli that uses the coordination of a heme tag to an L-histidine-immobilized sepharose (HIS) resin. This approach provides an affinity purification tag visible to the eye, facilitating tracking of the protein. We show that azurin and maltose binding protein are readily purified from cell lysate using the heme tag and HIS resin. Mild conditions are used; heme-tagged proteins are bound to the HIS resin in phosphate buffer, pH 7.0, and eluted by adding 200-500 mM imidazole or binding buffer at pH 5 or 8. The HIS resin exhibits a low level of nonspecific binding of untagged cellular proteins for the systems studied here. An additional advantage of the heme tag-HIS method for purification is that the heme tag can be used for protein quantification by using the pyridine hemochrome absorbance method for heme concentration determination.
Asher
Wesley B
WB
Department of Chemistry, University of Rochester, Rochester, New York 14627-0216, USA.
Bren
Kara L
KL
D000595:Amino Acid Sequence; D001400:Azurin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001667:Binding, Competitive; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006418:Heme / Q000031:analogs & derivatives / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D058619:Maltose-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012117:Resins, Synthetic; D012685:Sepharose; D013053:Spectrophotometry / Q000379:methods
D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D012117:Resins, Synthetic; D001400:Azurin; C023647:pyridine hemochrome; D006418:Heme; D006639:Histidine; D012685:Sepharose