Segregational Instability in<i>E. Coli</i>of Expression Plasmids Carrying Human Interferon Gamma Gene and its 3’-End Truncated Variants

Popov, M.; Petrov, S. V.; Kirilov, Kiril; Nacheva, Genoveva; Ivanov, Iván

doi:10.1080/13102818.2009.10818553

Cited by 12 publications

(6 citation statements)

References 5 publications

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“…Our assumption is supported by the fact that the initiation of the chromosomal DNA replication in E. coli is dependent on its transcriptional activity [45] where a direct interaction between DnaA (a bacterial replication initiator protein) and RNA polymerase has been found. In addition, we have previously shown that the segregation of the expression plasmid pP1SD-hIFNγ strongly depends on the level of hFNγ-mRNA in E. coli cells [46,47].…”

Section: Discussionmentioning

confidence: 99%

Nucleic acids in inclusion bodies obtained from E. coli cells expressing human interferon-gamma

2020

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Background: Inclusion bodies (IBs) are protein aggregates in recombinant bacterial cells containing mainly the target recombinant protein. Although it has been shown that IBs contain functional proteins along with protein aggregates, their direct application as pharmaceuticals is hindered by their heterogeneity and hazardous contaminants with bacterial origin. Therefore, together with the production of soluble species, IBs remain the main source for manufacture of recombinant proteins with medical application. The quality and composition of the IBs affect the refolding yield and further purification of the recombinant protein. The knowledge whether nucleic acids are genuine components or concomitant impurities of the IBs is a prerequisite for the understanding of the IBs formation and for development of optimized protocols for recombinant protein refolding and purification. IBs isolated from Escherichia coli overexpressing human interferon-gamma (hIFNγ), a protein with therapeutic application, were used as a model. Results: IBs were isolated from E. coli LE392 cells transformed with a hIFNγ expressing plasmid under standard conditions and further purified by centrifugation on a sucrose cushion, followed by several steps of sonication and washings with non-denaturing concentrations of urea. The efficiency of the purification was estimated by SDS-PAGE gel electrophoresis and parallel microbiological testing for the presence of residual intact bacteria. Phenol/chloroform extraction showed that the highly purified IBs contain both DNA and RNA. The latter were studied by UV spectroscopy and agarose gel electrophoresis combined with enzymatic treatment and hybridization. DNA was observed as a diffuse fraction mainly in the range of 250 to 1000 bp. RNA isolated by TRIzol ® also demonstrated a substantial molecular heterogeneity. Hybridization with 32 P-labelled oligonucleotides showed that the IBs contain rRNA and are enriched of hIFNγ mRNA. Conclusions: The results presented in this study indicate that the nucleic acids might be intrinsic components rather than co-precipitated impurities in the IBs. We assume that the nucleic acids are active participants in the aggregation of recombinant proteins and formation of the IBs that originate from the transcription and translation machinery of the microbial cell factory. Further studies are needed to ascertain this notion.

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Section: Discussionmentioning

confidence: 99%

Nucleic acids in inclusion bodies obtained from E. coli cells expressing human interferon-gamma

2020

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“…Subsequent transformation protocols based on conjugation or electroporation were then developed for C. sporogenes that were highly successful [ 21 , 23 ], allowing for tumour-specific expression of a transgene following colonisation by Clostridium . However, plasmid-based expression of genes can result in segregational instability, in which the proportion of plasmid-containing cells within a population decreases over multiple cycles of replication [ 24 , 25 ].…”

Section: Methods For Genetic Modification Of Clostridiummentioning

confidence: 99%

Clostridium Bacteria: Harnessing Tumour Necrosis for Targeted Gene Delivery

Theys,

Patterson,

Mowday

2024

Mol Diagn Ther

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Necrosis is a common feature of solid tumours that offers a unique opportunity for targeted cancer therapy as it is absent from normal healthy tissues. Tumour necrosis provides an ideal environment for germination of the anaerobic bacterium Clostridium from endospores, resulting in tumour-specific colonisation. Two main species, Clostridium novyi -NT and Clostridium sporogenes , are at the forefront of this therapy, showing promise in preclinical models. However, anti-tumour activity is modest when used as a single agent, encouraging development of Clostridium as a tumour-selective gene delivery system. Various methods, such as allele-coupled exchange and CRISPR–cas9 technology, can facilitate the genetic modification of Clostridium , allowing chromosomal integration of transgenes to ensure long-term stability of expression. Strains of Clostridium can be engineered to express prodrug-activating enzymes, resulting in the generation of active drug selectively in the tumour microenvironment (a concept termed Clostridium -directed enzyme prodrug therapy). More recently, Clostridium strains have been investigated in the context of cancer immunotherapy, either in combination with immune checkpoint inhibitors or with engineered strains expressing immunomodulatory molecules such as IL-2 and TNF-α. Localised expression of these molecules using tumour-targeting Clostridium strains has the potential to improve delivery and reduce systemic toxicity. In summary, Clostridium species represent a promising platform for cancer therapy, with potential for localised gene delivery and immunomodulation selectively within the tumour microenvironment. The ongoing clinical progress being made with C. novyi -NT, in addition to developments in genetic modification techniques and non-invasive imaging capabilities, are expected to further progress Clostridium as an option for cancer treatment.

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“…E. coli, as a well-characterized cell factory, represents the workhorse of choice for accomplishing controlled production of a variety of products due to its ease of reprogramming using synthetic biology tools and its robust bioprocessing capabilities. , Major concerns regarding the use of engineered gene circuits are related to the metabolic responses associated with recombinant expression, such as growth inhibition and protein synthesis leading to plasmid instability. , These responses result in heterogeneous cell populations in which differential growth of plasmid-bearing and plasmid-free cells occurs . Regarding BC biosynthesis, only a few studies have been published on mathematical modeling of naturally synthesized BC, which mainly concentrated on empirical microbial growth, glucose consumption, and substrate mass transfer prediction ignoring the regulatory mechanisms involved. , …”

Section: Introductionmentioning

confidence: 99%

“…28,29 These responses result in heterogeneous cell populations in which differential growth of plasmid-bearing and plasmid-free cells occurs. 30 Regarding BC biosynthesis, only a few studies have been published on mathematical modeling of naturally synthesized BC, which mainly concentrated on empirical microbial growth, glucose consumption, and substrate mass transfer prediction ignoring the regulatory mechanisms involved. 31,32 Herein, the rational modeling approach developed by Tsipa et al (2018) 18 was applied to the gene circuit of cellulose biosynthesis in the E. coli biosystem.…”

Section: Introductionmentioning

confidence: 99%

Linking Engineered Gene Circuit Kinetic Modeling to Cellulose Biosynthesis Prediction in Escherichia coli: Toward Bioprocessing of Microbial Cell Factories

Buldum

Tsipa

Mantalaris

2020

Ind. Eng. Chem. Res.

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Microbial cell factories synthesize value-added products; their bioprocessing with the aid of synthetic and system biology represents a green and sustainable alternative to the traditional chemical industry. Currently, bioprocess performance prediction of microbial cell factories is limited. Herein, we present a rational modeling approach linking the designed engineered gene circuit to bioprocess kinetics, whereby the engineered gene circuit model informs the formulation of product biosynthesis and is coupled to microbial growth. We achieve dynamic gene expression and estimation of enzyme synthesis of the gene circuit. Significantly, this modeling approach considers plasmid stability, which may decrease the productivity of recombinant systems. We demonstrate the validity of the approach using a bacterial cellulose (BC) biosynthesis cell factory in Escherichia coli. This kinetic model is a practical and complementary approach to systems and synthetic biology for the robust operation of microbial cell factory systems and their bioprocess applications.

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Segregational Instability inE. Coliof Expression Plasmids Carrying Human Interferon Gamma Gene and its 3’-End Truncated Variants

Cited by 12 publications

References 5 publications

Nucleic acids in inclusion bodies obtained from E. coli cells expressing human interferon-gamma

Nucleic acids in inclusion bodies obtained from E. coli cells expressing human interferon-gamma

Clostridium Bacteria: Harnessing Tumour Necrosis for Targeted Gene Delivery

Linking Engineered Gene Circuit Kinetic Modeling to Cellulose Biosynthesis Prediction in Escherichia coli: Toward Bioprocessing of Microbial Cell Factories

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