title: Remarkable alkaline stability of an engineered protein A as immunoglobulin affinity ligand: C domain having only one amino acid substitution.
abstract: Protein A affinity chromatography is the standard purification process for the capture of therapeutic antibodies. The individual IgG-binding domains of protein A (E, D, A, B, C) have highly homologous amino acid sequences. From a previous report, it has been assumed that the C domain has superior resistance to alkaline conditions compared to the other domains. We investigated several properties of the C domain as an IgG-Fc capture ligand. Based on cleavage site analysis of a recombinant protein A using a protein sequencer, the C domain was found to be the only domain to have neither of the potential alkaline cleavage sites. Circular dichroism (CD) analysis also indicated that the C domain has good physicochemical stability. Additionally, we evaluated the amino acid substitutions at the Gly-29 position of the C domain, as the Z domain (an artificial B domain) acquired alkaline resistance through a G29A mutation. The G29A mutation proved to increase the alkaline resistance of the C domain, based on BIACORE analysis, although the improvement was significantly smaller than that observed for the B domain. Interestingly, a number of other amino acid mutations at the same position increased alkaline resistance more than did the G29A mutation. This result supports the notion that even a single mutation on the originally alkali-stable C domain would improve its alkaline stability. An engineered protein A based on this C domain is expected to show remarkable performance as an affinity ligand for immunoglobulin.
authors.affiliation: Frontier Biochemical and Medical Research Laboratories, Kaneka Corp., 1-8 Miyamae-cho, Takasago-cho, Takasago, Hyogo, 676-8688, Japan.
authors.forename: Kazunobu
authors.initials: K
authors.lastname: Minakuchi
authors.forename: Dai
authors.initials: D
authors.lastname: Murata
authors.forename: Yuji
authors.initials: Y
authors.lastname: Okubo
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Nakano
authors.forename: Shinichi
authors.initials: S
authors.lastname: Yoshida
mesh_terms: D000468:Alkalies / Q000737:chemistry*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D005998:Glycine / Q000737:chemistry / Q000235:genetics; D007136:Immunoglobulins / Q000737:chemistry*; D008024:Ligands; D008969:Molecular Sequence Data; D015202:Protein Engineering; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013205:Staphylococcal Protein A / Q000737:chemistry*
chemical_list: D000468:Alkalies; D007136:Immunoglobulins; D008024:Ligands; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; D005998:Glycine
keywords: IgG-binding domain; affinity ligand; physicochemical stability; protein engineering; therapeutic antibody