Protein engineering of an IgG-binding domain allows milder elution conditions during affinity chromatography

One of the problems in the recovery of antibodies by affinity chromatograph y is the low pH, which is normally essential to elute the bound material fr om the column. Here, we have addressed this problem by constructing destabi lized mutants of a domain analogue (domain Z) from an IgG-binding bacterial receptor, protein A. In ol-der to destabilize the IgG-binding domain, two protein engineered variants were constructed using site-directed mutagenesi s of the second loop of this antiparallel three-helix bundle domain. In the first mutant (Z6C), the second loop was extended with six glycines in orde r to evaluate the significance of the loop length. In the second mutant (ZL 4G), the original loop sequence was exchanged for glycines in order to eval uate the importance of the loop forming residues. Both mutated variants hav e a lower a-helical content, as well as a lower thermal and chemical stabil ity compared to the parent 2-molecule. The affinity to IgG was slightly low ered in both cases, mainly due to higher dissociation rates. Interestingly, the elution studies showed that most of the bound IgG-molecules could be e luted at a pH as high as 4.5 from columns with the engineered ligands, whil e only 70% of the bound IgG could be eluted from the matrix with the parent Z as ligand. (C) 2000 Elsevier Science B.V. All rights reserved.