Kinetically inert Co(III) linkage through an engineered metal binding site: Specific orientation of recombinant human papillomavirus type 16 E7 protein on a solid support

Abstract

We report that engineered metal binding sites, or chelating peptides (CPs), can be used to site-specifically orient and immobilize recombinant CP-proteins on a solid support by forming a kinetically inert ternary complex with immobilized iminodiacetato-Co(III). The specific orientation and kinetic inertness of the linkage provide many advantages over conventional immobilization techniques. The utility of this novel immobilization chemistry was demonstrated using recombinant E7 protein from human papillomavirus type 16 with a CP, Met-His-Trp-His-His-His, at its N-terminus. The immobilized kinetically inert Co(III) complex of CP-E7 served as an extremely robust affinity matrix for the single-step purification of anti-E7 antibodies from rabbit antiserum. In addition, naturally occurring proteins that bind immobilized Co(II), such as myoglobin, can also be converted to kinetically inert immobilized complexes.