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Per Aqueous Liquid Chromatography (PALC) as a Simple Method for Native Separation of Protein A

Aboul-Enein, Hassan Y., Rigi, Garshasb, Farhadpour, Mohsen, Ghasempour, Alireza, Ahmadian, Gholamreza
Chromatographia 2017 v.80 no.11 pp. 1633-1639
Western blotting, antibodies, biotechnology, cost effectiveness, enzyme-linked immunosorbent assay, histidine, hydrophilicity, liquid chromatography, medicine, pH, polyacrylamide gel electrophoresis, purification methods, recombinant proteins, salt concentration, silica gel, solvents
Staphylococcal protein A (protein A) is an important protein frequently used in research studies within the fields of biomedicine and biotechnology. Due to some limitations in available protein purification methods which can hold the native structure of the protein A without changing the folding or adding histidine to structure of this protein, its separation in the native form is difficult. In this study, a new cost-effective and powerful technique was introduced for separation of the full-length and truncated forms of recombinant protein A, without any alteration in their 3D structures. Per aqueous liquid chromatography with bare silica gel stationary phase and water:acetonitrile as the mobile phase was proved to be an attractive choice among the range of separation methods. Similar to hydrophilic liquid chromatography, this method employs high percentage of water in mobile phase. The effects of mobile phase composition, pH, and salt concentration on the retention behavior of protein A on bare silica gel stationary phases were investigated. In this method, applying high amounts of aqueous solvent accompanied by a minimum percentage of organic solvent could successfully separate protein A with preservation of folding, and any affinity-tagged group such as histidine has not occurred on its structure. Purity of the fractions obtained by the proposed method was confirmed using SDS-PAGE, western blotting, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. According to the results of ELISA, separated proteins retained their ability of binding to antibody.