Development of a Methodology to Adapt an Equilibrium Buffer/Wash Applied to the Purification of hGPN2 Protein Expressed in <i>Escherichia coli</i> Using an IMAC Immobilized Metal Affinity Chromatography System
Protein purification is a complex and non-standardized process; the fact that proteins have different structural types making it difficult to create a standard methodology to obtain them in a pure, soluble, and homogeneous form. The present study shows the selective development of a buffer suitable...
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MDPI AG
2022-06-01
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| Online Access: | https://www.mdpi.com/2297-8739/9/7/164 |
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| author | Jorge Juárez-Lucero María del Rayo Guevara-Villa Anabel Sánchez-Sánchez Raquel Díaz-Hernández Leopoldo Altamirano-Robles |
| author_facet | Jorge Juárez-Lucero María del Rayo Guevara-Villa Anabel Sánchez-Sánchez Raquel Díaz-Hernández Leopoldo Altamirano-Robles |
| author_sort | Jorge Juárez-Lucero |
| collection | DOAJ |
| description | Protein purification is a complex and non-standardized process; the fact that proteins have different structural types making it difficult to create a standard methodology to obtain them in a pure, soluble, and homogeneous form. The present study shows the selective development of a buffer suitable for proteins of interest that allows high concentrations of hGPN2 protein to be obtained with low polydispersion and high homogeneity and purity. By taking the different reagents used in the construction of different buffers as a basis and performing purifications using different additives in different concentrations to determine the optimal amounts, the developed process helps to minimize the bonds, maintain solubility, release the proteins present in inclusion bodies, and provide an adequate environment for obtaining high concentrations of pure protein. GPN proteins are of unknown function, have not been purified in high concentrations, and have been found as part of the RNA polymerase assembly; if they are not expressed, the cell dies, and overexpression of certain GPN proteins has been linked to decreased survival in patients with invasive ductal carcinoma breast cancer types ER+ and HER2+. The results of the present study show that the use of the buffer developed for recombinant hGPN2 protein expressed in <i>Escherichia coli</i> could be manipulated in order to isolate the protein in a totally pure form and without the use of protease inhibitor tablets. The resulting homogeneity and low polydispersion was corroborated by studies carried out using dynamic dispersion analysis. Thanks to these properties, it can be used for crystallography or structural genomics studies. |
| first_indexed | 2024-03-09T10:12:56Z |
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| id | doaj.art-dd28390f4e204f129734fd904da380c9 |
| institution | Directory Open Access Journal |
| issn | 2297-8739 |
| language | English |
| last_indexed | 2024-03-09T10:12:56Z |
| publishDate | 2022-06-01 |
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| record_format | Article |
| series | Separations |
| spelling | doaj.art-dd28390f4e204f129734fd904da380c92023-12-01T22:41:15ZengMDPI AGSeparations2297-87392022-06-019716410.3390/separations9070164Development of a Methodology to Adapt an Equilibrium Buffer/Wash Applied to the Purification of hGPN2 Protein Expressed in <i>Escherichia coli</i> Using an IMAC Immobilized Metal Affinity Chromatography SystemJorge Juárez-Lucero0María del Rayo Guevara-Villa1Anabel Sánchez-Sánchez2Raquel Díaz-Hernández3Leopoldo Altamirano-Robles4Instituto Nacional de Astrofísica, Óptica y Electrónica, Tonantzintla, Puebla 72840, MexicoLaboratorio de Adsorción y Cromatografía, Centro de Química del Instituto de Ciencias de la Benemérita Universidad Autónoma de Puebla, Puebla 72000, MexicoInstituto Nacional de Astrofísica, Óptica y Electrónica, Tonantzintla, Puebla 72840, MexicoInstituto Nacional de Astrofísica, Óptica y Electrónica, Tonantzintla, Puebla 72840, MexicoInstituto Nacional de Astrofísica, Óptica y Electrónica, Tonantzintla, Puebla 72840, MexicoProtein purification is a complex and non-standardized process; the fact that proteins have different structural types making it difficult to create a standard methodology to obtain them in a pure, soluble, and homogeneous form. The present study shows the selective development of a buffer suitable for proteins of interest that allows high concentrations of hGPN2 protein to be obtained with low polydispersion and high homogeneity and purity. By taking the different reagents used in the construction of different buffers as a basis and performing purifications using different additives in different concentrations to determine the optimal amounts, the developed process helps to minimize the bonds, maintain solubility, release the proteins present in inclusion bodies, and provide an adequate environment for obtaining high concentrations of pure protein. GPN proteins are of unknown function, have not been purified in high concentrations, and have been found as part of the RNA polymerase assembly; if they are not expressed, the cell dies, and overexpression of certain GPN proteins has been linked to decreased survival in patients with invasive ductal carcinoma breast cancer types ER+ and HER2+. The results of the present study show that the use of the buffer developed for recombinant hGPN2 protein expressed in <i>Escherichia coli</i> could be manipulated in order to isolate the protein in a totally pure form and without the use of protease inhibitor tablets. The resulting homogeneity and low polydispersion was corroborated by studies carried out using dynamic dispersion analysis. Thanks to these properties, it can be used for crystallography or structural genomics studies.https://www.mdpi.com/2297-8739/9/7/164protein purificationhGPN2 proteinIMAC chromatography |
| spellingShingle | Jorge Juárez-Lucero María del Rayo Guevara-Villa Anabel Sánchez-Sánchez Raquel Díaz-Hernández Leopoldo Altamirano-Robles Development of a Methodology to Adapt an Equilibrium Buffer/Wash Applied to the Purification of hGPN2 Protein Expressed in <i>Escherichia coli</i> Using an IMAC Immobilized Metal Affinity Chromatography System Separations protein purification hGPN2 protein IMAC chromatography |
| title | Development of a Methodology to Adapt an Equilibrium Buffer/Wash Applied to the Purification of hGPN2 Protein Expressed in <i>Escherichia coli</i> Using an IMAC Immobilized Metal Affinity Chromatography System |
| title_full | Development of a Methodology to Adapt an Equilibrium Buffer/Wash Applied to the Purification of hGPN2 Protein Expressed in <i>Escherichia coli</i> Using an IMAC Immobilized Metal Affinity Chromatography System |
| title_fullStr | Development of a Methodology to Adapt an Equilibrium Buffer/Wash Applied to the Purification of hGPN2 Protein Expressed in <i>Escherichia coli</i> Using an IMAC Immobilized Metal Affinity Chromatography System |
| title_full_unstemmed | Development of a Methodology to Adapt an Equilibrium Buffer/Wash Applied to the Purification of hGPN2 Protein Expressed in <i>Escherichia coli</i> Using an IMAC Immobilized Metal Affinity Chromatography System |
| title_short | Development of a Methodology to Adapt an Equilibrium Buffer/Wash Applied to the Purification of hGPN2 Protein Expressed in <i>Escherichia coli</i> Using an IMAC Immobilized Metal Affinity Chromatography System |
| title_sort | development of a methodology to adapt an equilibrium buffer wash applied to the purification of hgpn2 protein expressed in i escherichia coli i using an imac immobilized metal affinity chromatography system |
| topic | protein purification hGPN2 protein IMAC chromatography |
| url | https://www.mdpi.com/2297-8739/9/7/164 |
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