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Author Spotlight: Optimizing Affinity Chromatography for His-Tagged FEN1 Protein
Published on: April 26, 2024
Molecular modification of Protein A to improve the elution pH and alkali resistance in affinity chromatography
Hai-Feng Xia1, Zhen-Dong Liang, Sha-Li Wang
1The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China, hfxia@jiangnan.edu.cn.
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.
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