A protocol for cloning, expression, and purification of Lysine exporter (LysE) gene of Mycobacterium tuberculosis [version 3; peer review: 2 approved]
Background Tuberculosis (TB) is among the deadliest diseases and a significant cause of illnessacross the globe. Several studies on mycobacterial proteins, such as proteases and transporters that are essential for survival and pathogenesis have aimed to develop an efficient anti-tubercular agent. In...
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F1000 Research Ltd
2024-02-01
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| Online Access: | https://f1000research.com/articles/12-297/v3 |
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| author | Supriya Kashikar Zahiruddin Syed Quazi Vinod Agarkar Shilpa Upadhyay Archana Dhok |
| author_facet | Supriya Kashikar Zahiruddin Syed Quazi Vinod Agarkar Shilpa Upadhyay Archana Dhok |
| author_sort | Supriya Kashikar |
| collection | DOAJ |
| description | Background Tuberculosis (TB) is among the deadliest diseases and a significant cause of illnessacross the globe. Several studies on mycobacterial proteins, such as proteases and transporters that are essential for survival and pathogenesis have aimed to develop an efficient anti-tubercular agent. In mycobacterium, lysine exporter (LysE) is an amino acid transporter and a probable target for an anti-tubercular agent as it is responsible for bacterial growth inhibition and is also absent in the widely used Bacillus Calmette-Guérin (BCG) vaccine. Methods Some studies have purified LysE using different protocols. This study describes a protocol for purifying different constructs of LysE, focusing on its hydrophobic region using immobilized metal affinity chromatography (IMAC) after expressing LysE gene in a bacterial expression system. pET vector (pET28a) is used as an expression vector. Amplified LysE gene is ligated with the pET28a vector, and the resultant plasmid is then transformed into E. coli cells. The vector has a histidine tag that makes the purification process convenient. After IMAC, the samples will be subjected to size-exclusion chromatography for further purification. Results Cloning and amplification findings will be analyzed using 1% agarose gel, and protein expression and purification outcomes will be examined using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Domain-specific constructs of LysE can be further analyzed as an anti-tubercular agent. Conclusions Despite being a potential anti-tubercular target, research is quite limited on this protein. Therefore, we aim to purify LysE protein for further analysis. Similar protocols have already been implemented to purify several other bacterial proteins with >95% purity. |
| first_indexed | 2024-03-08T05:15:36Z |
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| id | doaj.art-eb995714de5845dca4eeb5f6828f2bab |
| institution | Directory Open Access Journal |
| issn | 2046-1402 |
| language | English |
| last_indexed | 2024-03-08T05:15:36Z |
| publishDate | 2024-02-01 |
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| spelling | doaj.art-eb995714de5845dca4eeb5f6828f2bab2024-02-07T01:00:07ZengF1000 Research LtdF1000Research2046-14022024-02-0112161874A protocol for cloning, expression, and purification of Lysine exporter (LysE) gene of Mycobacterium tuberculosis [version 3; peer review: 2 approved]Supriya Kashikar0Zahiruddin Syed Quazi1https://orcid.org/0000-0001-5875-8277Vinod Agarkar2Shilpa Upadhyay3https://orcid.org/0000-0001-6258-5794Archana Dhok4CEO, GeNext Genomics Private Limited, Nagpur, Maharashtra, 440010, IndiaDirector, One Health Centre, Global Consortium for Public Health and Research (GCPHR), Department of Community Medicine, Jawaharlal Nehru Medical College, Datta Meghe Institute of Higher Education and Research, Wardha, Maharashtra, 442004, IndiaDirector, Research and Development, GeNext Genomics Private Limited, Nagpur, Maharashtra, 440010, IndiaResearch Associate, Division of Evidence Synthesis, Global Evidence Synthesis Initative (GESI), Datta Meghe Institute of Higher Education and Research, Wardha, Maharashtra, 442001, IndiaProfessor and Head, i-Health Consortium, Department of Biochemistry, Jawaharlal Nehru Medical College, Datta Meghe Institute of Higher Education and Research, Wardha, Maharashtra, 442004, IndiaBackground Tuberculosis (TB) is among the deadliest diseases and a significant cause of illnessacross the globe. Several studies on mycobacterial proteins, such as proteases and transporters that are essential for survival and pathogenesis have aimed to develop an efficient anti-tubercular agent. In mycobacterium, lysine exporter (LysE) is an amino acid transporter and a probable target for an anti-tubercular agent as it is responsible for bacterial growth inhibition and is also absent in the widely used Bacillus Calmette-Guérin (BCG) vaccine. Methods Some studies have purified LysE using different protocols. This study describes a protocol for purifying different constructs of LysE, focusing on its hydrophobic region using immobilized metal affinity chromatography (IMAC) after expressing LysE gene in a bacterial expression system. pET vector (pET28a) is used as an expression vector. Amplified LysE gene is ligated with the pET28a vector, and the resultant plasmid is then transformed into E. coli cells. The vector has a histidine tag that makes the purification process convenient. After IMAC, the samples will be subjected to size-exclusion chromatography for further purification. Results Cloning and amplification findings will be analyzed using 1% agarose gel, and protein expression and purification outcomes will be examined using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Domain-specific constructs of LysE can be further analyzed as an anti-tubercular agent. Conclusions Despite being a potential anti-tubercular target, research is quite limited on this protein. Therefore, we aim to purify LysE protein for further analysis. Similar protocols have already been implemented to purify several other bacterial proteins with >95% purity.https://f1000research.com/articles/12-297/v3Lysine exporter (LysE) Amino acid transporter Anti-tubercular agent Expression and purification Immobilized Metal-Affinity Chromatography (IMAC) Histidine-tageng |
| spellingShingle | Supriya Kashikar Zahiruddin Syed Quazi Vinod Agarkar Shilpa Upadhyay Archana Dhok A protocol for cloning, expression, and purification of Lysine exporter (LysE) gene of Mycobacterium tuberculosis [version 3; peer review: 2 approved] F1000Research Lysine exporter (LysE) Amino acid transporter Anti-tubercular agent Expression and purification Immobilized Metal-Affinity Chromatography (IMAC) Histidine-tag eng |
| title | A protocol for cloning, expression, and purification of Lysine exporter (LysE) gene of Mycobacterium tuberculosis [version 3; peer review: 2 approved] |
| title_full | A protocol for cloning, expression, and purification of Lysine exporter (LysE) gene of Mycobacterium tuberculosis [version 3; peer review: 2 approved] |
| title_fullStr | A protocol for cloning, expression, and purification of Lysine exporter (LysE) gene of Mycobacterium tuberculosis [version 3; peer review: 2 approved] |
| title_full_unstemmed | A protocol for cloning, expression, and purification of Lysine exporter (LysE) gene of Mycobacterium tuberculosis [version 3; peer review: 2 approved] |
| title_short | A protocol for cloning, expression, and purification of Lysine exporter (LysE) gene of Mycobacterium tuberculosis [version 3; peer review: 2 approved] |
| title_sort | protocol for cloning expression and purification of lysine exporter lyse gene of mycobacterium tuberculosis version 3 peer review 2 approved |
| topic | Lysine exporter (LysE) Amino acid transporter Anti-tubercular agent Expression and purification Immobilized Metal-Affinity Chromatography (IMAC) Histidine-tag eng |
| url | https://f1000research.com/articles/12-297/v3 |
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