Competitive Elution of Protein A Fusion Proteins Allows Specific Recovery Under Mild Conditions

Abstract

A novel system is described for mild elution of fusion proteins by competitive elution. The approach is based on displacement of immobilized fusions containing a monovalent IgG-binding staphylococcal protein A fragment (Z) from an IgG-affinity matrix by a divalent fragment fused to a serum-albumin-binding region derived from streptococcal protein G. Using real-time interaction analysis, the binding (K_aff) to polyclonal human IgG was found to be 3.3 (±0.4)×10⁸M^-1 for divalent ZZ and 2.0 (±0.1)×10⁷M^-1 for monovalent Z. This more than tenfold difference in binding strength ensures a high efficiency in the elution step. The competitor protein can specifically be removed and recovered from the elution mixture by subsequent passage through a human serum albumin(HSA)-affinity column, leaving only the target fusion protein in the flow-through fraction. Here, we show that a recombinant Klenow fragment of DNA polymerase I expressed in Escherichia coli can be recovered with high yield, and retained activity, from a crude bacterial lysate by IgG-affinity chromatography using mild conditions during both binding and elution.