Purification of Antibody Light Chains by Metal Affinity and Protein L Chromatography

Abstract

Immobilized metal affinity chromatography (IMAC), introduced in 1975 (1), relies on the formation of coordinate bonds between metal ions immobilized on a suitable support and electron donor groups in proteins. A polyhistidine tag (five or six His residues) placed at either the C- or N-terminus of a recombinant protein can form a stable chelate with immobilized transition metals. This allows fractionation of the target protein to 90–95% purity levels in a single chromatographic step (2–4). Metals like Cu²⁺ and Ni²⁺ bind surface-accessible His residues selectively. The high affinity of polyhistidine tags for commercially available metal resins (K _d 10⁻¹³ M or more) allows the use of stringent conditions to remove loosely bound proteins, while retaining the recombinant protein bound to the immobilized metal.