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High-Level Production of Soluble Cross-Reacting Material 197 in Escherichia coli Cytoplasm Due to Fine Tuning of the Target Gene’s mRNA Structure
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...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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MDPI
2023
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9844443/ https://www.ncbi.nlm.nih.gov/pubmed/36648835 http://dx.doi.org/10.3390/biotech12010009 |
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author | Khodak, Yulia Alexandrovna Ryazanova, Alexandra Yurievna Vorobiev, Ivan Ivanovich Kovalchuk, Alexander Leonidovich Ovechko, Nikolay Nikolaevich Aparin, Petr Gennadievich |
author_facet | Khodak, Yulia Alexandrovna Ryazanova, Alexandra Yurievna Vorobiev, Ivan Ivanovich Kovalchuk, Alexander Leonidovich Ovechko, Nikolay Nikolaevich Aparin, Petr Gennadievich |
author_sort | Khodak, Yulia Alexandrovna |
collection | PubMed |
description | 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—an additional Met [−1]. 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 150–270 mg/L (1.1–2.0 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′ area may significantly improve the target protein expression rate. |
format | Online Article Text |
id | pubmed-9844443 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-98444432023-01-18 High-Level Production of Soluble Cross-Reacting Material 197 in Escherichia coli Cytoplasm Due to Fine Tuning of the Target Gene’s mRNA Structure Khodak, Yulia Alexandrovna Ryazanova, Alexandra Yurievna Vorobiev, Ivan Ivanovich Kovalchuk, Alexander Leonidovich Ovechko, Nikolay Nikolaevich Aparin, Petr Gennadievich BioTech (Basel) Article 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—an additional Met [−1]. 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 150–270 mg/L (1.1–2.0 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′ area may significantly improve the target protein expression rate. MDPI 2023-01-11 /pmc/articles/PMC9844443/ /pubmed/36648835 http://dx.doi.org/10.3390/biotech12010009 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Khodak, Yulia Alexandrovna Ryazanova, Alexandra Yurievna Vorobiev, Ivan Ivanovich Kovalchuk, Alexander Leonidovich Ovechko, Nikolay Nikolaevich Aparin, Petr Gennadievich High-Level Production of Soluble Cross-Reacting Material 197 in Escherichia coli Cytoplasm Due to Fine Tuning of the Target Gene’s mRNA Structure |
title | High-Level Production of Soluble Cross-Reacting Material 197 in Escherichia coli Cytoplasm Due to Fine Tuning of the Target Gene’s mRNA Structure |
title_full | High-Level Production of Soluble Cross-Reacting Material 197 in Escherichia coli Cytoplasm Due to Fine Tuning of the Target Gene’s mRNA Structure |
title_fullStr | High-Level Production of Soluble Cross-Reacting Material 197 in Escherichia coli Cytoplasm Due to Fine Tuning of the Target Gene’s mRNA Structure |
title_full_unstemmed | High-Level Production of Soluble Cross-Reacting Material 197 in Escherichia coli Cytoplasm Due to Fine Tuning of the Target Gene’s mRNA Structure |
title_short | High-Level Production of Soluble Cross-Reacting Material 197 in Escherichia coli Cytoplasm Due to Fine Tuning of the Target Gene’s mRNA Structure |
title_sort | high-level production of soluble cross-reacting material 197 in escherichia coli cytoplasm due to fine tuning of the target gene’s mrna structure |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9844443/ https://www.ncbi.nlm.nih.gov/pubmed/36648835 http://dx.doi.org/10.3390/biotech12010009 |
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