title: Protein engineering of an IgG-binding domain allows milder elution conditions during affinity chromatography.
abstract: One of the problems in the recovery of antibodies by affinity chromatography is the low pH, which is normally essential to elute the bound material from the column. Here, we have addressed this problem by constructing destabilized mutants of a domain analogue (domain Z) from an IgG-binding bacterial receptor, protein A. In order to destabilize the IgG-binding domain, two protein engineered variants were constructed using site-directed mutagenesis of the second loop of this antiparallel three-helix bundle domain. In the first mutant (Z6G), the second loop was extended with six glycines in order to evaluate the significance of the loop length. In the second mutant (ZL4G), the original loop sequence was exchanged for glycines in order to evaluate the importance of the loop forming residues. Both mutated variants have a lower alpha-helical content, as well as a lower thermal and chemical stability compared to the parent Z-molecule. The affinity to IgG was slightly lowered in both cases, mainly due to higher dissociation rates. Interestingly, the elution studies showed that most of the bound IgG-molecules could be eluted at a pH as high as 4.5 from columns with the engineered ligands, while only 70% of the bound IgG could be eluted from the matrix with the parent Z as ligand.
authors.affiliation: Department of Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
authors.forename: S
authors.initials: S
authors.lastname: Gülich
authors.forename: M
authors.initials: M
authors.lastname: Uhlén
authors.forename: S
authors.initials: S
authors.lastname: Hober
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity / Q000379:methods*; D002942:Circular Dichroism; D007074:Immunoglobulin G / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013205:Staphylococcal Protein A / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D007074:Immunoglobulin G; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A