Remarkable alkaline stability of an engineered protein A as immunoglobulin affinity ligand: C domain having only one amino acid substitution. (6th August 2013)

• Record Type: Journal Article
• Authors: Minakuchi, Kazunobu; Murata, Dai; Okubo, Yuji; Nakano, Yoshiyuki; Yoshida, Shinichi
• Is Part Of: Protein science. Volume 22:Number 9(2013:Sep.)
• Page Start: 1230
• Page End: 1238
• Publication Date: 2013-08-06
• Subjects: Proteins -- Periodicals; 572.6
• DOI: 10.1002/pro.2310
• Languages: English
• ISSNs: 0961-8368

Abstract

<abstract abstract-type="main"> <title>Abstract</title> <p>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.</p> </abstract>