Production and purification of recombinant bovine osteopontin using Escherichia coli as a Cell Factory
Animal reproduction and improvement programs require the optimization of biotechnological tools capable of favoring reproductive rates in various species. The use of protein additives that improve sperm cryopreservation and in vitro embryo production seems to be an interesting alternative. Osteopont...
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| Format: | Online |
| Language: | spa |
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Universidad Pedagógica y Tecnológica de Colombia
2022
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| Online Access: | https://revistas.uptc.edu.co/index.php/ciencia_agricultura/article/view/14071 |
| _version_ | 1801706482618597376 |
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| author | Brijaldo Villamizar, Angela Patricia Londoño-Méndez, María Camila Arbeláez Ramírez, Luis Fernando Rueda, Fabian |
| author_facet | Brijaldo Villamizar, Angela Patricia Londoño-Méndez, María Camila Arbeláez Ramírez, Luis Fernando Rueda, Fabian |
| author_sort | Brijaldo Villamizar, Angela Patricia |
| collection | OJS |
| description | Animal reproduction and improvement programs require the optimization of biotechnological tools capable of favoring reproductive rates in various species. The use of protein additives that improve sperm cryopreservation and in vitro embryo production seems to be an interesting alternative. Osteopontin has been related to the fertilizing potential of the sperm and early embryonic development. The objective of this work was to determine the optimal conditions to produce recombinant Osteopontin (rOPN) by using Escherichia coli as a cell factory. For this, the OPN gene was inserted into an expression vector pET28(a+) inducible by IPTG, with resistance to Kanamycin and a histidine tail (6xHis-tag). The resulting construct was used to transform competent E. coli BL21-Star ™ cells. The transformed colonies were used to produce rOPN-H6 at 20, 30, and 37 °C, testing two concentrations of the inducer IPTG (1.0 and 0.1mM). A purification of rOPN-H6 was performed using imidazole affinity columns (10, 50, 200, 350, 500mM). The results showed that the production of rOPN-H6 was only successful at 37°C regardless of the concentration of IPTG used. Purification of rOPN-H6 was successful using imidazole at 200mM, with an apparent tendency to dimerization after obtaining purified protein. In this way, the best conditions to obtain recombinant OPN is concluded, suggesting its potential use in sperm cryopreservation assays and culture media for in vitro embryo production. |
| format | Online |
| id | oai:oai.revistas.uptc.edu.co:article-14071 |
| institution | Revista Ciencia y Agricultura |
| language | spa |
| publishDate | 2022 |
| publisher | Universidad Pedagógica y Tecnológica de Colombia |
| record_format | ojs |
| spelling | oai:oai.revistas.uptc.edu.co:article-140712022-11-30T00:13:48Z Production and purification of recombinant bovine osteopontin using Escherichia coli as a Cell Factory Producción Y Purificación De Osteopontina Bovina Recombinante Mediante Escherichia coli Como Fábrica Celular Brijaldo Villamizar, Angela Patricia Londoño-Méndez, María Camila Arbeláez Ramírez, Luis Fernando Rueda, Fabian Bovine Reproduction Cell Factories Seminal Plasma Sperm Cryopreservation Recombinant Proteins Reproducción Bovina Proteínas Recombinantes Fábricas Celulares Plasma Seminal Criopreservación De Semen Animal reproduction and improvement programs require the optimization of biotechnological tools capable of favoring reproductive rates in various species. The use of protein additives that improve sperm cryopreservation and in vitro embryo production seems to be an interesting alternative. Osteopontin has been related to the fertilizing potential of the sperm and early embryonic development. The objective of this work was to determine the optimal conditions to produce recombinant Osteopontin (rOPN) by using Escherichia coli as a cell factory. For this, the OPN gene was inserted into an expression vector pET28(a+) inducible by IPTG, with resistance to Kanamycin and a histidine tail (6xHis-tag). The resulting construct was used to transform competent E. coli BL21-Star ™ cells. The transformed colonies were used to produce rOPN-H6 at 20, 30, and 37 °C, testing two concentrations of the inducer IPTG (1.0 and 0.1mM). A purification of rOPN-H6 was performed using imidazole affinity columns (10, 50, 200, 350, 500mM). The results showed that the production of rOPN-H6 was only successful at 37°C regardless of the concentration of IPTG used. Purification of rOPN-H6 was successful using imidazole at 200mM, with an apparent tendency to dimerization after obtaining purified protein. In this way, the best conditions to obtain recombinant OPN is concluded, suggesting its potential use in sperm cryopreservation assays and culture media for in vitro embryo production. Los programas de reproducción y mejoramiento animal requieren la optimización de herramientas biotecnológicas capaces de favorecer los índices reproductivos en diversas especies. El uso de aditivos proteicos que mejoren la criopreservación espermática y la producción de embriones in vitro, parece ser una alternativa interesante. La Osteopontina se ha relacionado con el potencial fecundante del espermatozoide y con el desarrollo embrionario temprano. El objetivo de este trabajo fue determinar las condiciones óptimas para la producción de Osteopontina recombinante (rOPN) mediante el uso de Escherichia coli como fábrica celular. Para esto, el gen de la OPN se insertó en un vector de expresión pET28(a+) inducible por IPTG, con resistencia a la Kanamicina y una cola de histidinas (6xHis-tag). El constructo resultante se usó para transformar células competentes de E. Coli BL21-Star TM. Las colonias transformadas se usaron para la producción de rOPN-H6 a 20, 30 y 37 °C, probándose dos concentraciones del inductor IPTG (1.0 y 0.1mM). Se realizó una purificación de la rOPN-H6 mediante columnas de afinidad con imidazol (10, 50, 200, 350, 500mM). Los resultados evidenciaron que la producción de rOPN-H6 solo fue exitosa a 37°C independiente de la concentración de IPTG empleada. La purificación de la rOPN-H6 fue exitosa usando imidazol a 200mM, con una aparente tendencia a la dimerización luego de obtener la proteína purificada. De este modo, se concluye cuáles son las mejores condiciones para obtener la OPN recombinante, sugiriendo su potencial uso en ensayos de criopreservación espermática y en medios de cultivo para producción de embriones in vitro. Universidad Pedagógica y Tecnológica de Colombia 2022-07-07 info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion texto application/pdf text/xml https://revistas.uptc.edu.co/index.php/ciencia_agricultura/article/view/14071 10.19053/01228420.v19.n2.2022.14071 Ciencia y Agricultura; Vol. 19 No. 2 (2022): Mayo-Agosto Ciencia y Agricultura; Vol. 19 Núm. 2 (2022): Mayo-Agosto 2539-0899 spa https://revistas.uptc.edu.co/index.php/ciencia_agricultura/article/view/14071/11768 https://revistas.uptc.edu.co/index.php/ciencia_agricultura/article/view/14071/12386 Copyright (c) 2022 Fabian Rueda http://creativecommons.org/licenses/by/4.0 |
| spellingShingle | Bovine Reproduction Cell Factories Seminal Plasma Sperm Cryopreservation Recombinant Proteins Reproducción Bovina Proteínas Recombinantes Fábricas Celulares Plasma Seminal Criopreservación De Semen Brijaldo Villamizar, Angela Patricia Londoño-Méndez, María Camila Arbeláez Ramírez, Luis Fernando Rueda, Fabian Production and purification of recombinant bovine osteopontin using Escherichia coli as a Cell Factory |
| title | Production and purification of recombinant bovine osteopontin using Escherichia coli as a Cell Factory |
| title_alt | Producción Y Purificación De Osteopontina Bovina Recombinante Mediante Escherichia coli Como Fábrica Celular |
| title_full | Production and purification of recombinant bovine osteopontin using Escherichia coli as a Cell Factory |
| title_fullStr | Production and purification of recombinant bovine osteopontin using Escherichia coli as a Cell Factory |
| title_full_unstemmed | Production and purification of recombinant bovine osteopontin using Escherichia coli as a Cell Factory |
| title_short | Production and purification of recombinant bovine osteopontin using Escherichia coli as a Cell Factory |
| title_sort | production and purification of recombinant bovine osteopontin using escherichia coli as a cell factory |
| topic | Bovine Reproduction Cell Factories Seminal Plasma Sperm Cryopreservation Recombinant Proteins Reproducción Bovina Proteínas Recombinantes Fábricas Celulares Plasma Seminal Criopreservación De Semen |
| topic_facet | Bovine Reproduction Cell Factories Seminal Plasma Sperm Cryopreservation Recombinant Proteins Reproducción Bovina Proteínas Recombinantes Fábricas Celulares Plasma Seminal Criopreservación De Semen |
| url | https://revistas.uptc.edu.co/index.php/ciencia_agricultura/article/view/14071 |
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