A nickel chelate microtiter plate assay for six histidine-containing proteins.
Protein purification has been made significantly easier by the use of affinity tags that can be genetically engineered at either the amino- or carboxyl-terminus of recombinant proteins. One of the most widely used tags is six consecutive histidine residues or 6His tag. These residues bind with high affinity to metal ions immobilized on chelating resins even in the presence of denaturing agents and can be mildly eluted with imidazole. We report the methodology for the immobilization of six histidine-containing proteins onto microtiter plates. A derivative of nitrilotriacetic acid (NTA) was prepared. This derivative, N,N-bis[carboxymethyl]lysine (BCML), was easily coupled to a maleic anhydride-activated polystyrene microtiter plate. The plate was then charged with Ni2+ for the capture of the 6His-tagged proteins. Using two different recombinant proteins with the 6His tag at either the N- or C-terminus, we demonstrated that the binding to the Ni(2+)-NTA plate was specific for six histidine-containing proteins. Proteins lacking the 6His tags did not bind to the plate. The plate was used in a modified enzyme-linked immunoabsorbent assay format to quantitate protein concentrations and determine the affinity of protein-ligand interactions. The technology can also be extended to include high-throughput screening assays for antagonists of protein-protein interactions.
Paborsky
L R
LR
Gilead Sciences, Inc., Foster City, California 94404, USA.
Dunn
K E
KE
Gibbs
C S
CS
Dougherty
J P
JP
D000911:Antibodies, Monoclonal; D002614:Chelating Agents; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006639:Histidine / Q000032:analysis*; D007202:Indicators and Reagents; D016209:Interleukin-8 / Q000032:analysis; D008024:Ligands; D008239:Lysine / Q000031:analogs & derivatives / Q000138:chemical synthesis; D009532:Nickel; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives*; D009942:Organometallic Compounds; D011506:Proteins / Q000032:analysis*; D011994:Recombinant Proteins / Q000032:analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016324:Sequence Tagged Sites; D014168:Transferrin / Q000032:analysis
C097388:2-N,N-bis(carboxymethyl)lysine; D000911:Antibodies, Monoclonal; D002614:Chelating Agents; D007202:Indicators and Reagents; D016209:Interleukin-8; D008024:Ligands; D009942:Organometallic Compounds; D011506:Proteins; D011994:Recombinant Proteins; D014168:Transferrin; C088321:nickel nitrilotriacetic acid; D006639:Histidine; D009532:Nickel; D008239:Lysine; D009571:Nitrilotriacetic Acid