{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,16]],"date-time":"2026-01-16T16:59:19Z","timestamp":1768582759149,"version":"3.49.0"},"reference-count":55,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2023,1,11]],"date-time":"2023-01-11T00:00:00Z","timestamp":1673395200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"ATVD-TEAM Co., Ltd. (Moscow, Russia)"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["BioTech"],"abstract":"<jats:p>Cross-reacting material 197 (CRM197) is a non-toxic mutant of the diphtheria toxin and is widely used as a carrier protein in conjugate vaccines. This protein was first obtained from the supernatant of the mutant Corynebacterium diphtheriae strain. This pathogenic bacteria strain is characterized by a slow growth rate and a relatively low target protein yield, resulting in high production costs for CRM197. Many attempts have been made to establish high-yield protocols for the heterologous expression of recombinant CRM197 in different host organisms. In the present work, a novel CRM197-producing Escherichia coli strain was constructed. The target protein was expressed in the cytoplasm of SHuffle T7 E. coli cells without any additional tags and with a single potential mutation\u2014an additional Met [\u22121]. The fine tuning of the mRNA structure (the disruption of the single hairpin in the start codon area) was sufficient to increase the CRM197 expression level several times, resulting in 150\u2013270 mg\/L (1.1\u20132.0 mg\/g wet biomass) yields of pure CRM197 protein. Besides the high yield, the advantages of the obtained expression system include the absence of the necessity of CRM197 refolding or tag removal. Thus, an extensive analysis of the mRNA structure and the removal of the unwanted hairpins in the 5\u2032 area may significantly improve the target protein expression rate.<\/jats:p>","DOI":"10.3390\/biotech12010009","type":"journal-article","created":{"date-parts":[[2023,1,11]],"date-time":"2023-01-11T04:59:58Z","timestamp":1673413198000},"page":"9","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["High-Level Production of Soluble Cross-Reacting Material 197 in Escherichia coli Cytoplasm Due to Fine Tuning of the Target Gene\u2019s mRNA Structure"],"prefix":"10.3390","volume":"12","author":[{"given":"Yulia Alexandrovna","family":"Khodak","sequence":"first","affiliation":[{"name":"Federal State Institution, Federal Research Centre \u201cFundamentals of Biotechnology\u201d, The Russian Academy of Sciences, 119071 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alexandra Yurievna","family":"Ryazanova","sequence":"additional","affiliation":[{"name":"Federal State Institution, Federal Research Centre \u201cFundamentals of Biotechnology\u201d, The Russian Academy of Sciences, 119071 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0064-4458","authenticated-orcid":false,"given":"Ivan Ivanovich","family":"Vorobiev","sequence":"additional","affiliation":[{"name":"Federal State Institution, Federal Research Centre \u201cFundamentals of Biotechnology\u201d, The Russian Academy of Sciences, 119071 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2761-0442","authenticated-orcid":false,"given":"Alexander Leonidovich","family":"Kovalchuk","sequence":"additional","affiliation":[{"name":"National Research Center, Institute of Immunology Federal Medical-Biological Agency of Russia, 115522 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nikolay Nikolaevich","family":"Ovechko","sequence":"additional","affiliation":[{"name":"National Research Center, Institute of Immunology Federal Medical-Biological Agency of Russia, 115522 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Petr Gennadievich","family":"Aparin","sequence":"additional","affiliation":[{"name":"National Research Center, Institute of Immunology Federal Medical-Biological Agency of Russia, 115522 Moscow, Russia"},{"name":"ATVD-TEAM Co., Ltd., 115522 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,1,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"3838","DOI":"10.1016\/S0021-9258(19)43810-6","article-title":"Diphtheria toxin and related proteins. 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