title: Molecular modification of Protein A to improve the elution pH and alkali resistance in affinity chromatography.
abstract: Protein A of Staphylococcus aureus has been widely used as an affinity ligand for the purification of immunoglobulin. However, the low elution pH and the sensitivity to alkaline condition restricted the large-scale application of antibody purification. To overcome these disadvantages, the B domain was selected and mutated to Z domain and the recombinant Protein A was reconstructed by linking five Z domains. First, a section of six glycines was inserted into the second loop of Z domain, Z (6G). This increased the elution pH to 4.0-5.0. Then, the site-specific mutagenesis was conducted by replacing the 23rd asparagines to threonine and 30th phenylalanine to alanine, Z (N23T, F30A). These mutations made the recombinant Protein A shown a higher alkaline resistance than the nature Protein A. The work confirmed the modification of Protein A and exhibited the characteristics of recombinant Staphylococcal Protein A for antibody purification.
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China, hfxia@jiangnan.edu.cn.
authors.forename: Hai-Feng
authors.initials: HF
authors.lastname: Xia
authors.forename: Zhen-Dong
authors.initials: ZD
authors.lastname: Liang
authors.forename: Sha-Li
authors.initials: SL
authors.lastname: Wang
authors.forename: Pu-Qiang
authors.initials: PQ
authors.lastname: Wu
authors.forename: Xiong-Hua
authors.initials: XH
authors.lastname: Jin
mesh_terms: D000327:Adsorption; D000468:Alkalies / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000302:isolation & purification; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D015202:Protein Engineering / Q000379:methods*; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D013205:Staphylococcal Protein A / Q000737:chemistry* / Q000235:genetics*
chemical_list: D000468:Alkalies; D007074:Immunoglobulin G; D013205:Staphylococcal Protein A