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Affinity purification of human IgG using immobilised, mutated immunoglobulin-binding domains from protein A of Staphylococcus aureus.

Many pathogenic bacteria produce cell wall proteins that bind to host serum proteins such as immunoglobulins [l] and albumin [2]. These proteins are thought to help the bacteria evade the host immune system [3]. The common pathogen Staphylococcus aureus produces one such protein called protein A. This has five consecutive immunoglobulin (Ig) binding domains, each of about 58 amino acids residues, which interact strongly with the interface between the CH2 and CH3 domains of IgG-Fc. A weaker interaction also occurs between one of these domains and the VH domain of other classes of Ig [4]. This protein has therefore been used for many years for the affinity purification of Ig from serum. However, there are problems associated with the use of this protein in these procedures such as the requirement of a low pH to elute the bound Ig from the column, typically pH 2 3. We have been studymg the nature of the interactions between recombinant single domain proteins (SPA) based upon the B domain from protein A . Examination of the crystal structure of the complex formed between the B domain of protein A and human IgG-Fcl [5] shows that apart from four possible hydrogen bond interactions the predominant contribution to complex stability is from hydrophobic interactions. We have replaced For example, in the complex formed between IgG-Fc and SPA Asn32 forms a hydrogen bond contact with the amide group of Gln3 11 of IgG-Fc. A mutant bearing an Asn32His replacement was made and competitive ELISA experiments showed that the Kd for its complex with human IgG-Fcl at pH 8.0 is 45nM (Kd for non-mutated SPA = 40nM) whereas at pH 5.0 it increases approximately four fold to 10lnM. Figure 1 shows the elution profile of human IgG-Fcl from a column made from this mutant protein immobilised onto triazine-activated agarose. The bound IgG-Fcl was eluted from the column by a pH gradient made from 0.5M sodium acetate buffer pH 5.0 and 0.5M acetic acid pH 3.0. The profile shows that the majority of the applied human Fcl elutes at pH 5.0 rather than at pH 2 3 typical of the wild-type protein.