{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,28]],"date-time":"2026-08-28T06:18:17Z","timestamp":1787897897390,"version":"build-2784847793"},"reference-count":30,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,8,28]],"date-time":"2026-08-28T00:00:00Z","timestamp":1787875200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Bioeng. Biotechnol."],"abstract":"<jats:p>\n                    Extracellular protein expression in\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    is an elegant solution that addresses the complex issue of protein misfolding while simultaneously simplifying downstream processing steps. Human TNF-\u03b1 was chosen as the target protein for export since it is a therapeutically important cytokine. Different genomic knockouts were tested for the ability to sustain and enhance protein expression, and BW25113 \u0394(\n                    <jats:italic>elaA<\/jats:italic>\n                    +\n                    <jats:italic>cysW<\/jats:italic>\n                    ) knockout was found to give a sustained and high level of expression. To improve secretion, various tags were tested, and the MBP tag at the N-terminal end was found to give maximum enhancement in the export of hTNF-\u03b1. Even the linker peptide was found to play a critical role in export, with the Ek linker giving the highest extracellular secretion, while the intein sequence completely blocked export. The co-expression of pSecAB, which is involved in protein transport to the periplasm, was also found to be helpful in enhancing extracellular protein titers. Interestingly, pelB performed poorly as compared to the native signal sequence of MBP, which gave better results. Culture conditions were optimized, and it was observed that growing cells in TB medium at a temperature of 25\u00a0\u00b0C, coupled with a pulse of concentrated nutrients at 24\u00a0h, led to a very high extracellular accumulation of \u223c1.3\u00a0g\/L of MBP-hTNF-\u03b1 in shake flask culture. The protein was purified and tested using L929 cells for bioactivity. Thus, a combination of genomic and bioprocess strategies allowed us to obtain high levels of soluble and active extracellular expression of hTNF-\u03b1, making this a very attractive strategy for protein production.\n                  <\/jats:p>","DOI":"10.3389\/fbioe.2026.1897529","type":"journal-article","created":{"date-parts":[[2026,8,28]],"date-time":"2026-08-28T05:34:26Z","timestamp":1787895266000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Designing an optimized strategy for extracellular expression of recombinant human TNF-\u03b1 in Escherichia coli"],"prefix":"10.3389","volume":"14","author":[{"family":"Akanksha","sequence":"first","affiliation":[{"name":"University School of Biotechnology, Guru Gobind Singh Indraprastha University","place":["Dwarka, New Delhi, India"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sounak","family":"Dasgupta","sequence":"additional","affiliation":[{"name":"Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi","place":["Hauz Khas, New Delhi, India"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Priyanka","family":"Jain","sequence":"additional","affiliation":[{"name":"Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi","place":["Hauz Khas, New Delhi, India"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Manisha","family":"Kuanr","sequence":"additional","affiliation":[{"name":"Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi","place":["Hauz Khas, New Delhi, India"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gaurav","family":"Pandey","sequence":"additional","affiliation":[{"name":"University School of Biotechnology, Guru Gobind Singh Indraprastha University","place":["Dwarka, New Delhi, India"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Krishna Jyoti","family":"Mukherjee","sequence":"additional","affiliation":[{"name":"Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi","place":["Hauz Khas, New Delhi, India"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,8,28]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"120","DOI":"10.1007\/s10529-025-03655-8","article-title":"An effective strategy for soluble bovine enterokinase expression in Escherichia coli","volume":"47","author":"Akanksha","year":"2025","journal-title":"Biotechnol. 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