A NICKEL CHELATE MICROTITER PLATE ASSAY FOR 6 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 u sed tags is six consecutive histidine residues or 6His tag. These resi dues bind with high affinity to metal ions immobilized on chelating re sins even in the presence of denaturing agents and can be mildly elute d 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 wi th Ni2+ for the capture of the 6His-tagged proteins. Using two differe nt recombinant proteins with the 6His tag at either the N- or C-termin us, we demonstrated that the binding to the Ni2+-NTA plate was specifi c for six histidine-containing proteins. Proteins lacking the 6His tag s did not bind to the plate. The plate was used in a modified enzyme-l inked immunoabsorbent assay format to quantitate protein concentration s and determine the affinity of protein-ligand interactions. The techn ology can also be extended to include high-throughput screening assays for antagonists of protein-protein interactions. (C) 1996 Academic Pr ess, Inc.