Dataset Information

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: 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.

SUBMITTER: Khodak YA

PROVIDER: S-EPMC9844443 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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High-Level Production of Soluble Cross-Reacting Material 197 in <i>Escherichia coli</i> Cytoplasm Due to Fine Tuning of the Target Gene's mRNA Structure.

Khodak Yulia Alexandrovna YA Ryazanova Alexandra Yurievna AY Vorobiev Ivan Ivanovich II Kovalchuk Alexander Leonidovich AL Ovechko Nikolay Nikolaevich NN Aparin Petr Gennadievich PG

Biotech (Basel (Switzerland)) 20230111 1

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 <i>Corynebacterium diphtheriae</i> 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 ...[more]

PMID: 36648835

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Project description:To create a dataset of PSMs that does not exhibit tryptic bias, we selected PSMs with a uniform distribution of amino acids at the C-terminal peptide positions from two datasets: MassIVE-KB [Wang2018] and PROSPECT [Shouman2022]. The MassIVE-KB dataset contains 30 million PSMs and consists entirely of data generated using trypsin; hence, only a small proportion of the MassIVE-KB peptides do not end in K or R, corresponding to those that appear at the C-terminus of the corresponding protein. The PROSPECT dataset is a collection of 61 million PSMs generated from synthetic peptides. To avoid overrepresentation of some peptides in this dataset, we randomly selected at most 100 PSMs per unique peptide, similar to the processing that was done during the creation of the MassIVE-KB dataset. This pre-selection step reduced the size of the PROSPECT dataset to 12.6 million PSMs. Finally, to create a non-enzymatic dataset containing 1 million peptides, we selected 50,000 PSMs for each canonical amino acid. These PSMs were selected at random from MassIVE-KB, then supplemented as necessary from PROSPECT to obtain the desired count (see Yilmaz et al. [Yilmaz2023] Supplementary Table S1). This dataset contained PSMs from 247,859 unique peptides, which were randomly split into training, validation and test sets in the ratio 80/10/10. FTP directory contains the mgf files corresponding to train, validation and test splits. [Wang2018] M. Wang, J. Wang, J. Carver, B. Pullman, S. Won Cha, N. Bandeira, "Assembling the Community-Scale Discoverable Human Proteome", Cell Systems, Volume 7, Issue 4, 2018. [Shouman2022] O. Shouman, W. Gabriel, V. Giurcoiu, V. Sternlicht, M. Wilhelm, "PROSPECT: Labeled Tandem Mass Spectrometry Dataset for Machine Learning in Proteomics", NeurIPS Datasets and Benchmarks, 2022 [Yilmaz2023] M. Yilmaz*, W. Fondrie*, W. Bittremieux, R. Nelson, V. Ananth, S. Oh, and W. Noble,"Sequence-to-sequence translation from mass spectra to peptides with a transformer model", bioRxiv, 2023

Dataset Information

High-Level Production of Soluble Cross-Reacting Material 197 in Escherichia coli Cytoplasm Due to Fine Tuning of the Target Gene's mRNA Structure.

Publications

High-Level Production of Soluble Cross-Reacting Material 197 in <i>Escherichia coli</i> Cytoplasm Due to Fine Tuning of the Target Gene's mRNA Structure.

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