High-Level Production of Soluble Cross-Reacting Material 197 in Escherichia coli Cytoplasm Due to Fine Tuning of the Target Gene’s mRNA Structure
Abstract
:1. Introduction
2. Materials and Methods
2.1. Generation of the CRM197-Encoding Gene
2.2. Molecular Cloning
2.3. Mutagenesis
2.4. RNA Secondary Structure Analysis
2.5. Analytical Gene Expression
2.6. Kinetics of Gene Expression at Different Temperatures
2.7. Cell Cultivation in a Fermenter
2.8. Refolding of the Inclusion Body Fraction
2.9. Isolation of Periplasmic Proteins
2.10. CRM197 Purification
2.11. Laemmli Electrophoresis
2.12. Western Blotting
2.13. Native Gel Electrophoresis
2.14. Analytical Size-Exclusion Chromatography
2.15. N-Terminal Protein Sequencing
3. Results
3.1. Gene and Vector Construction
3.2. Refolding of CRM197 from Inclusion Bodies
3.3. Periplasmic Expression of CRM197
3.4. Choice of Expression Strain and Vector
3.5. Optimization of Expression Temperature
3.6. Mutagenesis of mRNA
3.7. Large-Scale CRM197 Expression and Purification
3.8. Analysis of the Purified CRM197
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Vaccine | Marketing Authorization Holder | Bacterial Polysaccharides | CRM197 Amount in One Dose, µg |
---|---|---|---|
Menveo | GSK Vaccines S.r.l. | Neisseria meningitidis polysaccharides, serogroups A, C, Y, and W-135 | 32.7–64.1 |
Menjugate | GSK Vaccines S.r.l. | Neisseria meningitidis polysaccharides, serogroup C (strain C11) | 12.5–25.0 |
Prevenar 13 | Pfizer Europe MA EEIG | Streptococcus pneumoniae polysaccharides, serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F | 32 |
Vaxneuvance | Merck Sharp & Dohme B.V. | Streptococcus pneumoniae polysaccharides, serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F | 30 |
Year | Expression Host | Compartmentalization | Solubility | Refolding Required | Protein Processing Required | Fermentation Scale | Cell Density | Protein Yield, mg/L | Reference |
---|---|---|---|---|---|---|---|---|---|
1983 | Corynebacterium diphtheriae | Secreted | Soluble | No | No | 5-L fermenter | Low | 175–250 3 | [10] |
1999 | Bacillus subtilis | Secreted | Soluble | No | No | Shake flask | Low | 7.1 3 | [21] |
2011 | Escherichia coli | Cytoplasmic | Insoluble (inclusion bodies) | Yes | Yes 1 | Shake flask | Low | 250 ± 50 4 | [11] |
2016 | E. coli | Cytoplasmic | Soluble + insoluble | No | Yes 1 | Shake flask | Low | 154 ± 13 3 | [12] |
2017 | E. coli | Periplasmic | Soluble (?) 5 | No | No | 150-L fermenter | High | 3200 4,5 | [14] |
2017 | E. coli | Cytoplasmic | Soluble + insoluble | No | No | 2-L fermenter | High | 106 ± 1.5 3 | [15] |
2018 | E. coli | Cytoplasmic | Insoluble (inclusion bodies) | Yes | Yes 1 | Shake flask | Low | 196 4 | [16] |
2018 | E. coli | Cytoplasmic | Insoluble (inclusion bodies) | Yes | No | 20-L fermenter | High | Not specified | [17] |
2021 | Pichia pastoris | Secreted | Soluble | No | No | 16-L bioreactor | High | 113 6 | [25] |
2022 | E. coli | Cytoplasmic | Soluble + insoluble | No | Yes 2 | Shake flask | Low | 130 4 | [18] |
2022 | E. coli | Cytoplasmic | Soluble | No | No | 16-L fermenter | High | 150–270 6 | This work |
Plasmid Name | Original Vector | Mutation Type | The Initial Codons of the Target Gene | The Initial Amino Acid Residues of the Target Protein |
---|---|---|---|---|
pET28a-CRM197-gcc | pET28a | None | ATGGGAGCCGACGAC | MGADD |
pHYP-CRM197-gcc | pHYP | None | ATGGGAGCCGACGAC | MGADD |
pHYP-CRM197-peri | pHYP | Leader peptide insertion | ATGATTAAATTTCTCTCTGCATTAATTCTTCTACTGGTCACGACGGCGGCTCAGGCTGGAGCCGACGAC | MIKFLSALILLLVTTAAQAGADD |
pHYP-D1 | pHYP | Deletion | ATG---GCCGACGAC | M-ADD |
pHYP-D2 | pHYP | Deletion | ATG------GACGAC | M--DD |
pHYP-D3 | pHYP | Deletion | ATG---------GAC | M---D |
pHYP-CRM197-gca | pHYP | Synonymous substitution | ATGGGAGCAGACGAC | MGADD |
pHYP-CRM197-gcg | pHYP | Synonymous substitution | ATGGGAGCGGACGAC | MGADD |
pHYP-CRM197-gct | pHYP | Synonymous substitution | ATGGGAGCTGACGAC | MGADD |
pHYP-CRM197-acc | pHYP | Nonsynonymous substitution | ATGGGAACCGACGAC | MGTDD |
pHYP-CRM197-tcc | pHYP | Nonsynonymous substitution | ATGGGATCCGACGAC | MGSDD |
pHYP-CRM197-ccc | pHYP | Nonsynonymous substitution | ATGGGACCCGACGAC | MGPDD |
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Khodak, Y.A.; Ryazanova, A.Y.; Vorobiev, I.I.; Kovalchuk, A.L.; Ovechko, N.N.; Aparin, P.G. High-Level Production of Soluble Cross-Reacting Material 197 in Escherichia coli Cytoplasm Due to Fine Tuning of the Target Gene’s mRNA Structure. BioTech 2023, 12, 9. https://doi.org/10.3390/biotech12010009
Khodak YA, Ryazanova AY, Vorobiev II, Kovalchuk AL, Ovechko NN, Aparin PG. High-Level Production of Soluble Cross-Reacting Material 197 in Escherichia coli Cytoplasm Due to Fine Tuning of the Target Gene’s mRNA Structure. BioTech. 2023; 12(1):9. https://doi.org/10.3390/biotech12010009
Chicago/Turabian StyleKhodak, Yulia Alexandrovna, Alexandra Yurievna Ryazanova, Ivan Ivanovich Vorobiev, Alexander Leonidovich Kovalchuk, Nikolay Nikolaevich Ovechko, and Petr Gennadievich Aparin. 2023. "High-Level Production of Soluble Cross-Reacting Material 197 in Escherichia coli Cytoplasm Due to Fine Tuning of the Target Gene’s mRNA Structure" BioTech 12, no. 1: 9. https://doi.org/10.3390/biotech12010009