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Molecular Modification of Protein A to Improve the Elution pH and Alkali Resistance in Affinity Chromatography

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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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Acknowledgments

This work was supported by the National High Technology Research and Development Program of China (863 Program, 2012AA021201), the National Natural Science Foundation of China (21206054), the Research Fund for the Doctoral Program of Higher Education of China (20110093120001), the 111 Project (No. 111-2-06), and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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Authors and Affiliations

  1. The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China

    Hai-Feng Xia, Zhen-Dong Liang, Sha-Li Wang & Pu-Qiang Wu

  2. National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China

    Hai-Feng Xia

  3. Zhejiang Hisun Pharmaceutical Co., Ltd, Taizhou, 318000, China

    Xiong-Hua Jin

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  1. Hai-Feng Xia
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Correspondence to Hai-Feng Xia.

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Xia, HF., Liang, ZD., Wang, SL. et al. Molecular Modification of Protein A to Improve the Elution pH and Alkali Resistance in Affinity Chromatography. Appl Biochem Biotechnol 172, 4002–4012 (2014). https://doi.org/10.1007/s12010-014-0818-1

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