{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,12]],"date-time":"2026-10-12T16:56:02Z","timestamp":1791824162826,"version":"4.3.4"},"reference-count":18,"publisher":"Wiley","issue":"10","license":[{"start":{"date-parts":[[2026,10,12]],"date-time":"2026-10-12T00:00:00Z","timestamp":1791763200000},"content-version":"vor","delay-in-days":11,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T00:00:00Z","timestamp":1790812800000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2026,10]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    Our previous immunoproteomic studies on patients with acute brucellosis identified outer membrane protein 25 (Omp25) of\n                    <jats:italic>Brucella melitensis<\/jats:italic>\n                    as one of the immunogenic antigens, indicating its potential to be used as a diagnostic marker and a tool to differentiate acute and chronic human brucellosis. Expression and purification of Omp25 are technically challenging using conventional approaches owing to its membrane association. Existing expression and extraction protocols frequently lead to host cell toxicity, low protein yields, and poor reproducibility. To address these limitations, we developed a modified approach for the expression and purification of recombinant Omp25 protein that is standardized and reproducible and yields protein efficiently. The truncated\n                    <jats:italic>omp25<\/jats:italic>\n                    gene is cloned and expressed in\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    BL21 (DE3) cells, and induction is carried out at 28\u00b0C for 6 hr. The expressed recombinant Omp25 protein is predominantly found in the insoluble fraction, so it is extracted under denaturing conditions, purified by Ni\u2010NTA affinity chromatography, refolded by stepwise dialysis, and desalted prior to characterization. In our hands, this set of protocols yielded 47.75 mg purified, high\u2010quality recombinant Omp25 protein per liter of bacterial culture. SDS\u2010PAGE analysis demonstrated a single predominant protein band at \u223c24.3 kDa with no detectable contaminating protein bands, indicating successful purification. recombinant Omp25\u2013based indirect ELISA confirmed that the refolded protein retained its antigenic properties, exhibiting significantly higher reactivity with sera from culture\u2010confirmed brucellosis patients than with healthy control sera (p &lt; 0.001). This optimized set of protocols provides a reproducible workflow for the production of highly purified recombinant Omp25 protein and offers quantitative performance metrics that facilitate reproducibility and support this protein's application in immunological and diagnostic studies. \u00a9 2026 The Author(s).\n                    <jats:italic>Current Protocols<\/jats:italic>\n                    published by Wiley Periodicals LLC.\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 1<\/jats:bold>\n                    : Expression and extraction of\n                    <jats:italic>Brucella melitensis<\/jats:italic>\n                    Omp25 protein from\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    BL21 (DE3) cells\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 2<\/jats:bold>\n                    : Purification of recombinant Omp25 protein\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 3<\/jats:bold>\n                    : Refolding and desalting of recombinant Omp25 protein\n                  <\/jats:p>","DOI":"10.1002\/cpz1.70481","type":"journal-article","created":{"date-parts":[[2026,10,12]],"date-time":"2026-10-12T16:39:46Z","timestamp":1791823186000},"source":"Crossref","is-referenced-by-count":0,"title":["Optimized Expression and Purification of Outer Membrane Protein 25 (Omp25) of\n                    <i>Brucella melitensis<\/i>"],"prefix":"10.1002","volume":"6","author":[{"given":"Harshita","family":"Mogaveera","sequence":"first","affiliation":[{"name":"Department of Microbiology, Kasturba Medical College Manipal Academy of Higher Education  Manipal India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sudipta","family":"Patra","sequence":"additional","affiliation":[{"name":"Department of Microbiology Sapthagiri Institute of Medical Sciences and Research Center  Bangalore India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Niladri Bhusan","family":"Pati","sequence":"additional","affiliation":[{"name":"Manipal Center for Biotherapeutics Research Manipal Academy of Higher Education  Manipal India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chiranjay","family":"Mukhopadhyay","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Kasturba Medical College Manipal Academy of Higher Education  Manipal India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Manjunath","family":"Joshi","sequence":"additional","affiliation":[{"name":"Department of Ageing Research, Manipal School of Life Sciences Manipal Academy of Higher Education  Manipal India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vandana Kalwaje","family":"Eshwara","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Kasturba Medical College Manipal Academy of Higher Education  Manipal India"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,10,12]]},"reference":[{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12917\u2010015\u20100587\u20102"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/s0076\u20106879(00)26058\u20108"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0076\u20106879(09)63017\u20102"},{"key":"e_1_2_8_5_1","first-page":"231","volume-title":"Methods in enzymology","author":"Duong\u2010Ly K. 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