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Microbial Cell Factories Mar 2024Escherichia coli is one of the most commonly used host organisms for the production of biopharmaceuticals, as it allows for cost-efficient and fast recombinant protein...
BACKGROUND
Escherichia coli is one of the most commonly used host organisms for the production of biopharmaceuticals, as it allows for cost-efficient and fast recombinant protein expression. However, challenging proteins are often produced with low titres or as inclusion bodies, and the manufacturing process needs to be developed individually for each protein. Recently, we developed the CASPON technology, a generic fusion tag-based platform process for high-titer soluble expression including a standardized downstream processing and highly specific enzymatic cleavage of the fusion tag. To assess potential strategies for further improvement of the N-terminally fused CASPON tag, we modified the 5'UTR and 5' region of the tag-coding mRNA to optimize the ribosome-mRNA interactions.
RESULTS
In the present work, we found that by modifying the 5'UTR sequence of a pET30acer plasmid-based system, expression of the fusion protein CASPON-tumour necrosis factor α was altered in laboratory-scale carbon-limited fed-batch cultivations, but no significant increase in expression titre was achieved. Translation efficiency was highest for a construct carrying an expression enhancer element and additionally possessing a very favourable interaction energy between ribosome and mRNA (∆G). However, a construct with comparatively low transcriptional efficiency, which lacked the expression enhancer sequence and carried the most favourable ∆G tested, led to the highest recombinant protein formation alongside the reference pET30a construct. Furthermore, we found, that by introducing synonymous mutations within the nucleotide sequence of the T7AC element of the CASPON tag, utilizing a combination of rare and non-rare codons, the free folding energy of the nucleotides at the 5' end (-4 to + 37) of the transcript encoding the CASPON tag increased by 6 kcal/mol. Surprisingly, this new T7AC variant led to improved recombinant protein titres by 1.3-fold up to 5.3-fold, shown with three industry-relevant proteins in lab-scale carbon limited fed-batch fermentations under industrially relevant conditions.
CONCLUSIONS
This study reveals some of the complex interdependencies between the ribosome and mRNA that govern recombinant protein expression. By modifying the 5'UTR to obtain an optimized interaction energy between the mRNA and the ribosome (ΔG), transcript levels were changed, highlighting the different translation efficiencies of individual transcripts. It was shown that the highest recombinant titre was not obtained by the construct with the most efficient translation but by a construct with a generally high transcript amount coupled with a favourable ΔG. Furthermore, an unexpectedly high potential to enhance expression by introducing silent mutations including multiple rare codons into the 5'end of the CAPON tag's mRNA was identified. Although the titres of the fusion proteins were dramatically increased, no formation of inclusion bodies or negative impact on cell growth was observed. We hypothesize that the drastic increase in titre is most likely caused by better ribosomal binding site accessibility. Our study, which demonstrates the influence of changes in ribosome-mRNA interactions on protein expression under industrially relevant production conditions, opens the door to the applicability of the new T7AC tag in biopharmaceutical industry using the CASPON platform process.
Topics: RNA, Messenger; 5' Untranslated Regions; Escherichia coli; Codon; Recombinant Proteins; Carbon; Recombinant Fusion Proteins
PubMed: 38509572
DOI: 10.1186/s12934-024-02350-z -
Nucleic Acids Research Apr 2024Long single-stranded DNA (ssDNA) is a versatile molecular reagent with applications including RNA-guided genome engineering and DNA nanotechnology, yet its production is...
Long single-stranded DNA (ssDNA) is a versatile molecular reagent with applications including RNA-guided genome engineering and DNA nanotechnology, yet its production is typically resource-intensive. We introduce a novel method utilizing an engineered Escherichia coli 'helper' strain and phagemid system that simplifies long ssDNA generation to a straightforward transformation and purification procedure. Our method obviates the need for helper plasmids and their associated contamination by integrating M13mp18 genes directly into the E. coli chromosome. We achieved ssDNA lengths ranging from 504 to 20 724 nt with titers up to 250 μg/l following alkaline lysis purification. The efficacy of our system was confirmed through its application in primary T-cell genome modifications and DNA origami folding. The reliability, scalability and ease of our approach promise to unlock new experimental applications requiring large quantities of long ssDNA.
Topics: DNA, Single-Stranded; Escherichia coli; Genetic Engineering; Plasmids
PubMed: 38499480
DOI: 10.1093/nar/gkae189 -
Journal of Alzheimer's Disease : JAD 2024A hypothesis of Alzheimer's disease etiology is proposed describing how cellular stress induces excessive polyamine synthesis and recycling which can disrupt nucleoli....
A hypothesis of Alzheimer's disease etiology is proposed describing how cellular stress induces excessive polyamine synthesis and recycling which can disrupt nucleoli. Polyamines are essential in nucleolar functions, such as RNA folding and ribonucleoprotein assembly. Changes in the nucleolar pool of anionic RNA and cationic polyamines acting as counterions can cause significant nucleolar dynamics. Polyamine synthesis reduces S-adenosylmethionine which, at low levels, triggers tau phosphorylation. Also, polyamine recycling reduces acetyl-CoA needed for acetylcholine, which is low in Alzheimer's disease. Extraordinary nucleolar expansion and/or contraction can disrupt epigenetic control in peri-nucleolar chromatin, such as chromosome 14 with the presenilin-1 gene; chromosome 21 with the amyloid precursor protein gene; chromosome 17 with the tau gene; chromosome 19 with the APOE4 gene; and the inactive X chromosome (Xi; aka "nucleolar satellite") with normally silent spermine synthase (polyamine synthesis) and spermidine/spermine-N1-acetyltransferase (polyamine recycling) alleles. Chromosomes 17, 19 and the Xi have high concentrations of Alu elements which can be transcribed by RNA polymerase III if positioned nucleosomes are displaced from the Alu elements. A sudden flood of Alu RNA transcripts can competitively bind nucleolin which is usually bound to Alu sequences in structural RNAs that stabilize the nucleolar heterochromatic shell. This Alu competition leads to loss of nucleolar integrity with leaking of nucleolar polyamines that cause aggregation of phosphorylated tau. The hypothesis was developed with key word searches (e.g., PubMed) using relevant terms (e.g., Alzheimer's, lupus, nucleolin) based on a systems biology approach and exploring autoimmune disease tautology, gaining synergistic insights from other diseases.
Topics: Humans; Polyamines; Alzheimer Disease; Cell Nucleolus; Autoimmune Diseases; RNA
PubMed: 38489184
DOI: 10.3233/JAD-231184 -
Stem Cell Research & Therapy Mar 2024Mesenchymal stem/stromal cells (MSCs) can regenerate tissues through engraftment and differentiation but also via paracrine signalling via extracellular vesicles (EVs)....
BACKGROUND
Mesenchymal stem/stromal cells (MSCs) can regenerate tissues through engraftment and differentiation but also via paracrine signalling via extracellular vesicles (EVs). Fetal-derived MSCs (fMSCs) have been shown, both in vitro and in animal studies, to be more efficient than adult MSC (aMSCs) in generating bone and muscle but the underlying reason for this difference has not yet been clearly elucidated. In this study, we aimed to systematically investigate the differences between fetal and adult MSCs and MSC-derived EVs at the phenotypic, RNA, and protein levels.
METHODS
We carried out a detailed and comparative characterization of culture-expanded fetal liver derived MSCs (fMSCs) and adult bone marrow derived MSCs (aMSCs) phenotypically, and the MSCs and MSC-derived EVs were analysed using transcriptomics and proteomics approaches with RNA Sequencing and Mass Spectrometry.
RESULTS
Fetal MSCs were smaller, exhibited increased proliferation and colony-forming capacity, delayed onset of senescence, and demonstrated superior osteoblast differentiation capability compared to their adult counterparts. Gene Ontology analysis revealed that fMSCs displayed upregulated gene sets such as "Positive regulation of stem cell populations", "Maintenance of stemness" and "Muscle cell development/contraction/Myogenesis" in comparison to aMSCs. Conversely, aMSCs displayed upregulated gene sets such as "Complement cascade", "Adipogenesis", "Extracellular matrix glycoproteins" and "Cellular metabolism", and on the protein level, "Epithelial cell differentiation" pathways. Signalling entropy analysis suggested that fMSCs exhibit higher signalling promiscuity and hence, higher potency than aMSCs. Gene ontology comparisons revealed that fetal MSC-derived EVs (fEVs) were enriched for "Collagen fibril organization", "Protein folding", and "Response to transforming growth factor beta" compared to adult MSC-derived EVs (aEVs), whereas no significant difference in protein expression in aEVs compared to fEVs could be detected.
CONCLUSIONS
This study provides detailed and systematic insight into the differences between fMSCs and aMSCs, and MSC-derived EVs. The key finding across phenotypic, transcriptomic and proteomic levels is that fMSCs exhibit higher potency than aMSCs, meaning they are in a more undifferentiated state. Additionally, fMSCs and fMSC-derived EVs may possess greater bone forming capacity compared to aMSCs. Therefore, using fMSCs may lead to better treatment efficacy, especially in musculoskeletal diseases.
Topics: Animals; Transcriptome; Proteomics; Mesenchymal Stem Cells; Gene Expression Profiling; Extracellular Vesicles
PubMed: 38475970
DOI: 10.1186/s13287-024-03683-7 -
International Journal of Molecular... Mar 2024Ribosomal proteins (r-proteins) are abundant, highly conserved, and multifaceted cellular proteins in all domains of life. Most r-proteins have RNA-binding properties... (Review)
Review
Ribosomal proteins (r-proteins) are abundant, highly conserved, and multifaceted cellular proteins in all domains of life. Most r-proteins have RNA-binding properties and can form protein-protein contacts. Bacterial r-proteins govern the co-transcriptional rRNA folding during ribosome assembly and participate in the formation of the ribosome functional sites, such as the mRNA-binding site, tRNA-binding sites, the peptidyl transferase center, and the protein exit tunnel. In addition to their primary role in a cell as integral components of the protein synthesis machinery, many r-proteins can function beyond the ribosome (the phenomenon known as moonlighting), acting either as individual regulatory proteins or in complexes with various cellular components. The extraribosomal activities of r-proteins have been studied over the decades. In the past decade, our understanding of r-protein functions has advanced significantly due to intensive studies on ribosomes and gene expression mechanisms not only in model bacteria like or but also in little-explored bacterial species from various phyla. The aim of this review is to update information on the multiple functions of r-proteins in bacteria.
Topics: Ribosomal Proteins; Bacterial Proteins; Ribosomes; Protein Biosynthesis; Bacteria; Escherichia coli; RNA, Ribosomal
PubMed: 38474204
DOI: 10.3390/ijms25052957 -
MBio Apr 2024Rotavirus (RV) replication takes place in the viroplasms, cytosolic inclusions that allow the synthesis of virus genome segments and their encapsidation in the core...
UNLABELLED
Rotavirus (RV) replication takes place in the viroplasms, cytosolic inclusions that allow the synthesis of virus genome segments and their encapsidation in the core shell, followed by the addition of the second layer of the virion. The viroplasms are composed of several viral proteins, including NSP5, which serves as the main building block. Microtubules, lipid droplets, and miRNA-7 are among the host components recruited in viroplasms. We investigated the interaction between RV proteins and host components of the viroplasms by performing a pull-down assay of lysates from RV-infected cells expressing NSP5-BiolD2. Subsequent tandem mass spectrometry identified all eight subunits of the tailless complex polypeptide I ring complex (TRiC), a cellular chaperonin responsible for folding at least 10% of the cytosolic proteins. Our confirmed findings reveal that TRiC is brought into viroplasms and wraps around newly formed double-layered particles. Chemical inhibition of TRiC and silencing of its subunits drastically reduced virus progeny production. Through direct RNA sequencing, we show that TRiC is critical for RV replication by controlling dsRNA genome segment synthesis, particularly negative-sense single-stranded RNA. Importantly, cryo-electron microscopy analysis shows that TRiC inhibition results in defective virus particles lacking genome segments and polymerase complex (VP1/VP3). Moreover, TRiC associates with VP2 and NSP5 but not with VP1. Also, VP2 is shown to be essential for recruiting TRiC in viroplasms and preserving their globular morphology. This study highlights the essential role of TRiC in viroplasm formation and in facilitating virion assembly during the RV life cycle.
IMPORTANCE
The replication of rotavirus takes place in cytosolic inclusions termed viroplasms. In these inclusions, the distinct 11 double-stranded RNA genome segments are co-packaged to complete a genome in newly generated virus particles. In this study, we show for the first time that the tailless complex polypeptide I ring complex (TRiC), a cellular chaperonin responsible for the folding of at least 10% of the cytosolic proteins, is a component of viroplasms and is required for the synthesis of the viral negative-sense single-stranded RNA. Specifically, TRiC associates with NSP5 and VP2, the cofactor involved in RNA replication. Our study adds a new component to the current model of rotavirus replication, where TRiC is recruited to viroplasms to assist replication.
Topics: Rotavirus; Viral Replication Compartments; Viral Nonstructural Proteins; Cryoelectron Microscopy; Virus Replication; RNA; Peptides
PubMed: 38470055
DOI: 10.1128/mbio.00499-24 -
Research Square Feb 2024The prediction of RNA secondary structures is essential for understanding its underlying principles and applications in diverse fields, including molecular diagnostics...
The prediction of RNA secondary structures is essential for understanding its underlying principles and applications in diverse fields, including molecular diagnostics and RNA-based therapeutic strategies. However, the complexity of the search space presents a challenge. This work proposes a Graph Convolutional Network (GCNfold) for predicting the RNA secondary structure. GCNfold considers an RNA sequence as graph-structured data and predicts posterior base-pairing probabilities given the prior base-pairing probabilities, calculated using McCaskill's partition function. The performance of GCNfold surpasses that of the state-of-the-art folding algorithms, as we have incorporated minimum free energy information into the richly parameterized network, enhancing its robustness in predicting non-homologous RNA secondary structures. A Symmetric Argmax Post-processing algorithm ensures that GCNfold formulates valid structures. To validate our algorithm, we applied it to the SARS-CoV-2 E gene and determined the secondary structure of the E-gene across the subgenera.
PubMed: 38464300
DOI: 10.21203/rs.3.rs-3798842/v1 -
BioRxiv : the Preprint Server For... Mar 2024Long single-stranded DNA (ssDNA) is a versatile molecular reagent with applications including RNA-guided genome engineering and DNA nanotechnology, yet its production is...
Long single-stranded DNA (ssDNA) is a versatile molecular reagent with applications including RNA-guided genome engineering and DNA nanotechnology, yet its production is typically resource-intensive. We introduce a novel method utilizing an engineered "helper" strain and phagemid system that simplifies long ssDNA generation to a straightforward transformation and purification procedure. Our method obviates the need for helper plasmids and their associated contamination by integrating M13mp18 genes directly into the chromosome. We achieved ssDNA lengths ranging from 504 to 20,724 nucleotides with titers up to 250 μg/L following alkaline-lysis purification. The efficacy of our system was confirmed through its application in primary T cell genome modifications and DNA origami folding. The reliability, scalability, and ease of our approach promises to unlock new experimental applications requiring large quantities of long ssDNA.
PubMed: 38464036
DOI: 10.1101/2024.02.27.582394 -
BMC Microbiology Mar 2024Copper-induced gene expression in Xanthomonas campestris pv. campestris (Xcc) is typically evaluated using targeted approaches involving qPCR. The global response to...
BACKGROUND
Copper-induced gene expression in Xanthomonas campestris pv. campestris (Xcc) is typically evaluated using targeted approaches involving qPCR. The global response to copper stress in Xcc and resistance to metal induced damage is not well understood. However, homologs of heavy metal efflux genes from the related Stenotrophomonas genus are found in Xanthomonas which suggests that metal related efflux may also be present.
METHODS AND RESULTS
Gene expression in Xcc strain BrA1 exposed to 0.8 mM CuSO.5HO for 15 minutes was captured using RNA-seq analysis. Changes in expression was noted for genes related to general stress responses and oxidoreductases, biofilm formation, protein folding chaperones, heat-shock proteins, membrane lipid profile, multiple drug and efflux (MDR) transporters, and DNA repair were documented. At this timepoint only the cohL (copper homeostasis/tolerance) gene was upregulated as well as a chromosomal czcCBA efflux operon. An additional screen up to 4 hrs using qPCR was conducted using a wider range of heavy metals. Target genes included a cop-containing heavy metal resistance island and putative metal efflux genes. Several efflux pumps, including a copper resistance associated homolog from S. maltophilia, were upregulated under toxic copper stress. However, these pumps were also upregulated in response to other toxic heavy metals. Additionally, the temporal expression of the coh and cop operons was also observed, demonstrating co-expression of tolerance responses and later activation of part of the cop operon.
CONCLUSIONS
Overall, initial transcriptional responses focused on combating oxidative stress, mitigating protein damage and potentially increasing resistance to heavy metals and other biocides. A putative copper responsive efflux gene and others which might play a role in broader heavy metal resistance were also identified. Furthermore, the expression patterns of the cop operon in conjunction with other copper responsive genes allowed for a better understanding of the fate of copper ions in Xanthomonas. This work provides useful evidence for further evaluating MDR and other efflux pumps in metal-specific homeostasis and tolerance phenotypes in the Xanthomonas genus. Furthermore, non-canonical copper tolerance and resistance efflux pumps were potentially identified. These findings have implications for interpreting MIC differences among strains with homologous copLAB resistance genes, understanding survival under copper stress, and resistance in disease management.
Topics: Copper; Xanthomonas campestris; Xanthomonas; Bacterial Proteins
PubMed: 38461228
DOI: 10.1186/s12866-024-03206-7 -
Scientific Reports Mar 2024Arsenic (As) is a significant contaminant whose unrestrained entrance into different ecosystems has created global concern. At the cellular level, As forms unsteady...
Isolation, characterization, identification, genomics and analyses of bioaccumulation and biosorption potential of two arsenic-resistant bacteria obtained from natural environments.
Arsenic (As) is a significant contaminant whose unrestrained entrance into different ecosystems has created global concern. At the cellular level, As forms unsteady intermediates with genetic materials and perturbs different metabolic processes and proper folding of proteins. This study was the first in this region to explore, isolate, screen systematically, and intensively characterize potent As-tolerant bacterial strains from natural environments near Raiganj town of Uttar Dinajpur, West Bengal. In this study, two potent Gram-negative bacterial strains with high tolerance to the poisonous form of As, i.e., As(III) and As(V), were obtained. Both the isolates were identified using biochemical tests and 16S rRNA gene sequencing. These bacteria oxidized toxic As(III) into less poisonous As(V) and depicted tolerance towards other heavy metals. Comparative metabolic profiling of the isolates in control and As-exposed conditions through Fourier-transform infrared spectroscopy showed metabolic adjustments to cope with As toxicity. The metal removal efficiency of the isolates at different pH showed that one of the isolates, KG1D, could remove As efficiently irrespective of changes in the media pH. In contrast, the efficiency of metal removal by PF14 was largely pH-dependent. The cell mass of both the isolates was also found to favourably adsorb As(III). Whole genome sequence analysis of the isolates depicted the presence of the arsRBC genes of the arsenic operon conferring resistance to As. Owing to their As(III) oxidizing potential, high As bioaccumulation, and tolerance to other heavy metals, these bacteria could be used to bioremediate and reclaim As-contaminated sites.
Topics: Arsenic; Bioaccumulation; RNA, Ribosomal, 16S; Ecosystem; Bacteria; Metals, Heavy; Biodegradation, Environmental; Genomics
PubMed: 38459150
DOI: 10.1038/s41598-024-56082-6