{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,31]],"date-time":"2025-12-31T10:19:42Z","timestamp":1767176382029,"version":"build-2238731810"},"update-to":[{"DOI":"10.12688\/f1000research.131768.1","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2024,2,5]],"date-time":"2024-02-05T00:00:00Z","timestamp":1707091200000}},{"DOI":"10.12688\/f1000research.131768.2","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2024,2,5]],"date-time":"2024-02-05T00:00:00Z","timestamp":1707091200000}}],"reference-count":12,"publisher":"F1000 Research Ltd","license":[{"start":{"date-parts":[[2024,2,5]],"date-time":"2024-02-05T00:00:00Z","timestamp":1707091200000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["f1000research.com"],"crossmark-restriction":false},"short-container-title":["F1000Res"],"abstract":"<ns3:p>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-Gu\u00e9rin (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 &gt;95% purity.<\/ns3:p>","DOI":"10.12688\/f1000research.131768.3","type":"journal-article","created":{"date-parts":[[2024,2,5]],"date-time":"2024-02-05T10:10:11Z","timestamp":1707127811000},"page":"297","update-policy":"https:\/\/doi.org\/10.12688\/f1000research.crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["A protocol for cloning, expression, and purification of Lysine exporter (LysE) gene of Mycobacterium tuberculosis"],"prefix":"10.12688","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6258-5794","authenticated-orcid":false,"given":"Shilpa","family":"Upadhyay","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Archana","family":"Dhok","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vinod","family":"Agarkar","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Supriya","family":"Kashikar","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5875-8277","authenticated-orcid":false,"given":"Zahiruddin Syed","family":"Quazi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"2560","published-online":{"date-parts":[[2024,2,5]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"158","DOI":"10.3389\/fcimb.2018.00158","article-title":"Mycobacterium tuberculosis: An Adaptable Pathogen Associated With Multiple Human Diseases.","volume":"8","author":"Q Chai","year":"2018","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref2","first-page":"6","article-title":"The Role of Mycobacterium tuberculosis Rv1986 and Rv3823c in Stimulating Humoral and Cell-Mediated Immune Responses.","volume":"1","author":"F Chen","year":"2018"},{"key":"ref3","doi-asserted-by":"publisher","first-page":"6996-7003","DOI":"10.1128\/IAI.70.12.6996-7003.2002","article-title":"Identification of Novel Mycobacterium tuberculosis Antigens with Potential as Diagnostic Reagents or Subunit Vaccine Candidates by Comparative Genomics.","volume":"70","author":"P Cockle","year":"2002","journal-title":"Infect. Immun."},{"key":"ref4","doi-asserted-by":"publisher","first-page":"201-213","DOI":"10.1016\/S1389-1723(01)80251-6","article-title":"The cell wall barrier of Corynebacterium glutamicum and amino acid efflux.","volume":"92","author":"L Eggeling","year":"2001","journal-title":"J. Biosci. Bioeng."},{"key":"ref5","doi-asserted-by":"publisher","first-page":"503a-504a","DOI":"10.1016\/j.bpj.2019.11.2777","article-title":"Purification and Biophysical Characterization of LysE Membrane Exporter From Mycobacterium tuberculosis in Lipodiscs Made of Native E. coli Membranes and Detergent.","volume":"118","author":"E Georgieva","year":"2020a","journal-title":"Biophys. J."},{"key":"ref6","doi-asserted-by":"publisher","first-page":"1-1","DOI":"10.1096\/fasebj.2020.34.s1.09164","article-title":"Expression, purification and initial characterization of LysE membrane exporter from Mycobacterium tuberculosis: Towards comprehensive functional and structural study.","volume":"34","author":"E Georgieva","year":"2020b","journal-title":"FASEB J."},{"key":"ref7","doi-asserted-by":"publisher","first-page":"e1001237","DOI":"10.1371\/journal.ppat.1001237","article-title":"Hypoxia Induces an Immunodominant Target of Tuberculosis Specific T Cells Absent from Common BCG Vaccines.","volume":"6","author":"H Gideon","year":"2010","journal-title":"PLoS Pathog."},{"key":"ref8","doi-asserted-by":"publisher","first-page":"265-274","DOI":"10.1007\/s00253-001-0869-4","article-title":"Bacterial amino acid transport proteins: occurrence, functions, and significance for biotechnological applications.","volume":"58","author":"R Kr\u00e4mer","year":"2002","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref9","doi-asserted-by":"publisher","first-page":"59-66","DOI":"10.1590\/S0034-89102007000800009","article-title":"Vacina BCG contra tuberculose: efeito protetor e pol\u00edticas de vacina\u00e7\u00e3o.","volume":"41","author":"S Pereira","year":"2007","journal-title":"Rev. Sa\u00fade P\u00fablica."},{"key":"ref10","first-page":"110-115","article-title":"Cloning, expression, and refolding of Nsuccinyl-l-l diaminopimelic acid desuccinylase (DapE) of Mycobacterium tuberculosis.","volume":"7","author":"C Umbarkar","year":"2019","journal-title":"IJLSR."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"19-30","DOI":"10.1385\/1-59259-147-7:019","article-title":"Isolation of DNA from Mycobacterium tubercolosis.","volume":"54","author":"P Van Helden","year":"2001","journal-title":"Methods Mol. 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Martin, Seren Hamsici, Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College Dublin, Dublin, 19 Jul 2023, version 2, indexed","URL":"https:\/\/f1000research.com\/articles\/12-297\/v2#referee-response-176987","order":0,"name":"referee-response-176987","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.5256\/f1000research.150461.r232751, Dr. Rahul Sharma, University of Virginia, Virginia, 25 Jan 2024, version 2, indexed","URL":"https:\/\/f1000research.com\/articles\/12-297\/v2#referee-response-232751","order":1,"name":"referee-response-232751","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"The author(s) declared that no grants were involved in supporting this work","order":2,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}