A modified metal-ion affinity chromatography procedure for the purification of histidine-tagged recombinant proteins expressed in Drosophila S2 cells.
We have developed a modified method of immobilized metal-ion affinity chromatography (IMAC) that can be used for the purification of histidine-tagged proteins from conditioned medium containing free copper ions. Classical methods of IMAC purification, using resins such as Ni-NTA, have proven inefficient for this type of purification and require multiple steps due to the interference of divalent copper ions with the binding of His-tagged protein to the charged resin. In contrast, this modified IMAC procedure, using chelating Sepharose instead of Ni-NTA, enables efficient purification from copper-containing medium in a single step. This method appears to rely upon a preferential interaction of protein-copper complexes with immobilized chelating resin. We have utilized this method to purify active, His-tagged murine interleukin 12 from the conditioned medium of Drosophila S2 cells coexpressing recombinant p40 and His-tagged p35 subunits and for the purification of the extracellular domain of the erythropoietin receptor. This method should be applicable to the purification of a wide variety of His-tagged fusion proteins expressed in Drosophila cells and in other systems where free metal ions are present.
Lehr
R V
RV
Department of Protein Biochemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA.
Elefante
L C
LC
Kikly
K K
KK
O'Brien
S P
SP
Kirkpatrick
R B
RB
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D002614:Chelating Agents; D002846:Chromatography, Affinity / Q000379:methods*; D003001:Cloning, Molecular; D019327:Copper Sulfate; D017077:Culture Media, Conditioned; D004330:Drosophila / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D006639:Histidine / Q000737:chemistry; D007371:Interferon-gamma / Q000378:metabolism; D018664:Interleukin-12 / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D015335:Molecular Probes; D010957:Plasmids / Q000235:genetics; D017467:Receptors, Erythropoietin / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013154:Spleen / Q000166:cytology / Q000378:metabolism
D002614:Chelating Agents; D017077:Culture Media, Conditioned; D015335:Molecular Probes; D017467:Receptors, Erythropoietin; D011993:Recombinant Fusion Proteins; D018664:Interleukin-12; D006639:Histidine; D007371:Interferon-gamma; D019327:Copper Sulfate