scholarly journals Cell-free prediction of protein expression costs for growing cells

2017 ◽  
Author(s):  
Olivier Borkowski ◽  
Carlos Bricio ◽  
Michaela Murgiano ◽  
Guy-Bart Stan ◽  
Tom Ellis
Keyword(s):  
Gene Expression ◽  
Host Cells ◽  
Translation Efficiency ◽  
Heterologous Proteins ◽  
E Coli ◽  
Multiple Processes ◽  
Significant Burden ◽  
The Cost ◽  
The Relationship

Translating heterologous proteins places significant burden on host cells, consuming expression resources leading to slower cell growth and productivity. Yet predicting the cost of protein production for any gene is a major challenge, as multiple processes and factors determine translation efficiency. Here, to enable prediction of the cost of gene expression in bacteria, we describe a standard cell-free lysate assay that determines the relationship betweenin vivoand cell-free measurements and γ, a relative measure of the resource consumption when a given protein is expressed. When combined with a computational model of translation, this enables prediction of thein vivoburden placed on growingE. colicells for a variety of proteins of different functions and lengths. Using this approach, we can predict the burden of expressing multigene operons of different designs and differentiate between the fraction of burden related to gene expression compared to action of a metabolic pathway.

scholarly journals Quantifying cellular capacity to identify gene expression designs with reduced burden

2014 ◽  
Author(s):  
Francesca Ceroni ◽  
Rhys J R Algar ◽  
Guy-Bart Stan ◽  
Tom Ellis
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Heterologous Gene Expression ◽  
Heterologous Gene ◽  
E Coli ◽  
Equivalent Expression ◽  
Significant Burden ◽  
Cellular Capacity ◽  
Identify Gene Expression

Heterologous gene expression can be a significant burden to cells, consuming resources and causing decreased growth and stability. We describe here anin vivomonitor that tracksE. colicapacity changes in real-time and can be used to assay the burden synthetic constructs and their parts impose. By measuring capacity, construct designs with reduced burden can be identified and shown to predictably outperform less efficient designs, despite having equivalent expression outputs.

Procaryotic Expression of Single-Chain Variable-Fragment (scFv) Antibodies: Secretion in L-Form Cells of Proteus mirabilis Leads to Active Product and Overcomes the Limitations of Periplasmic Expression in Escherichia coli

1998 ◽  
Vol 64 (12) ◽  
pp. 4862-4869 ◽  
Author(s):  
Jörg F. Rippmann ◽  
Michaela Klein ◽  
Christian Hoischen ◽  
Bodo Brocks ◽  
Wolfgang J. Rettig ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proteus Mirabilis ◽  
Binding Activity ◽  
Host Cells ◽  
Heterologous Proteins ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
E Coli ◽  
Active Product ◽  
Periplasmic Expression

ABSTRACT Recently it has been demonstrated that L-form cells ofProteus mirabilis (L VI), which lack a periplasmic compartment, can be efficiently used in the production and secretion of heterologous proteins. In search of novel expression systems for recombinant antibodies, we compared levels of single-chain variable-fragment (scFv) production in Escherichia coliJM109 and P. mirabilis L VI, which express four distinct scFvs of potential clinical interest that show differences in levels of expression and in their tendencies to form aggregates upon periplasmic expression. Production of all analyzed scFvs in E. coli was limited by the severe toxic effect of the heterologous product as indicated by inhibition of culture growth and the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. In contrast, the L-form cells exhibited nearly unlimited growth under the tested production conditions for all scFvs examined. Moreover, expression experiments with P. mirabilis L VI led to scFv concentrations in the range of 40 to 200 mg per liter of culture medium (corresponding to volume yields 33- to 160-fold higher than those with E. coli JM109), depending on the expressed antibody. In a translocation inhibition experiment the secretion of the scFv constructs was shown to be an active transport coupled to the signal cleavage. We suppose that this direct release of the newly synthesized product into a large volume of the growth medium favors folding into the native active structure. The limited aggregation of scFv observed in the P. mirabilis L VI supernatant (occurring in a first-order-kinetics manner) was found to be due to intrinsic features of the scFv and not related to the expression process of the host cells. The P. mirabilis L VI supernatant was found to be advantageous for scFv purification. A two-step chromatography procedure led to homogeneous scFv with high antigen binding activity as revealed from binding experiments with eukaryotic cells.

scholarly journals Characterization, modelling and mitigation of gene expression burden in mammalian cells

2019 ◽  
Author(s):  
T Frei ◽  
F Cella ◽  
F Tedeschi ◽  
J Gutierrez ◽  
GB Stan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Genome Engineering ◽  
Host Cells ◽  
Genetic Circuits ◽  
Circuit Performance ◽  
Resource Requirements ◽  
Synthetic Circuit ◽  
Resource Aware

AbstractDespite recent advances in genome engineering, the design of genetic circuits in mammalian cells is still painstakingly slow and fraught with inexplicable failures. Here we demonstrate that competition for limited transcriptional and translational resources dynamically couples otherwise independent co-expressed exogenous genes, leading to diminished performance and contributing to the divergence between intended and actual function. We also show that the expression of endogenous genes is likewise impacted when genetic payloads are expressed in the host cells. Guided by a resource-aware mathematical model and our experimental finding that post-transcriptional regulators have a large capacity for resource redistribution, we identify and engineer natural and synthetic miRNA-based incoherent feedforward loop (iFFL) circuits that mitigate gene expression burden. The implementation of these circuits features the novel use of endogenous miRNAs as integral components of the engineered iFFL device, a versatile hybrid design that allows burden mitigation to be achieved across different cell-lines with minimal resource requirements. This study establishes the foundations for context-aware prediction and improvement of in vivo synthetic circuit performance, paving the way towards more rational synthetic construct design in mammalian cells.

In Vivo Expression of Murine Platelet Glycoprotein Ibα

Blood ◽  
1998 ◽  
Vol 92 (2) ◽  
pp. 488-495 ◽  
Author(s):  
Hiroyuki Fujita ◽  
Yoshimi Hashimoto ◽  
Susan Russell ◽  
Barbara Zieger ◽  
Jerry Ware
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Luciferase Activity ◽  
Blood Platelet ◽  
Northern Analysis ◽  
Platelet Glycoprotein ◽  
Heterologous Proteins ◽  
In Vivo Evaluation

Abstract We have performed a systematic in vivo evaluation of gene expression for the glycoprotein (GP) Ibα subunit of the murine platelet adhesion receptor, GP Ib-IX-V. This study is warranted by in vitro observations of human GP Ibα expression in cells of nonhematopoietic lineage and reports of regulation of the GP Ibα gene by cytokines. However, an in vivo role for a GP Ib-IX-V receptor has not been established beyond that described for normal megakaryocyte/platelet physiology and hemostasis. Our Northern analysis of mouse organs showed high levels of GP Ibα mRNA in bone marrow with a similar expression pattern recapitulated in mice containing a luciferase transgene under the control of the murine GP Ibα promoter. Consistently high levels of luciferase activity were observed in the two hematopoietic organs of mice, bone marrow (1,400 relative light units/μg of protein [RLUs]) and spleen (500 RLUs). Reproducible, but low-levels of luciferase activity were observed in heart, aorta, and lung (30 to 60 RLUs). Among circulating blood cells, the luciferase activity was exclusively localized in platelets. No increase in GP Ibα mRNA or luciferase activity was observed after treatment of mice with lipopolysaccharides (LPS) or tumor necrosis factor-α (TNF-α). We conclude the murine GP Ibα promoter supports a high level of gene expression in megakaryocytes and can express heterologous proteins allowing an in vivo manipulation of platelet-specific proteins in the unique environment of a blood platelet.

scholarly journals In Vivo mRNA Profiling of Uropathogenic Escherichia coli from Diverse Phylogroups Reveals Common and Group-Specific Gene Expression Profiles

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Piotr Bielecki ◽  
Uthayakumar Muthukumarasamy ◽  
Denitsa Eckweiler ◽  
Agata Bielecka ◽  
Sarah Pohl ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Specific Gene ◽  
Content Type ◽  
E Coli ◽  
Human Host ◽  
Tract Infections

ABSTRACTmRNA profiling of pathogens during the course of human infections gives detailed information on the expression levels of relevant genes that drive pathogenicity and adaptation and at the same time allows for the delineation of phylogenetic relatedness of pathogens that cause specific diseases. In this study, we used mRNA sequencing to acquire information on the expression ofEscherichia colipathogenicity genes during urinary tract infections (UTI) in humans and to assign the UTI-associatedE. coliisolates to different phylogenetic groups. Whereas thein vivogene expression profiles of the majority of genes were conserved among 21E. colistrains in the urine of elderly patients suffering from an acute UTI, the specific gene expression profiles of the flexible genomes was diverse and reflected phylogenetic relationships. Furthermore, genes transcribedin vivorelative to laboratory media included well-described virulence factors, small regulatory RNAs, as well as genes not previously linked to bacterial virulence. Knowledge on relevant transcriptional responses that drive pathogenicity and adaptation of isolates to the human host might lead to the introduction of a virulence typing strategy into clinical microbiology, potentially facilitating management and prevention of the disease.IMPORTANCEUrinary tract infections (UTI) are very common; at least half of all women experience UTI, most of which are caused by pathogenicEscherichia colistrains. In this study, we applied massive parallel cDNA sequencing (RNA-seq) to provide unbiased, deep, and accurate insight into the nature and the dimension of the uropathogenicE. coligene expression profile during an acute UTI within the human host. This work was undertaken to identify key players in physiological adaptation processes and, hence, potential targets for new infection prevention and therapy interventions specifically aimed at sabotaging bacterial adaptation to the human host.

Comparison of Vibrio and Firefly Luciferases as Reporter Gene Systems for Use in Bacteria and Plants

1996 ◽  
Vol 23 (1) ◽  
pp. 75 ◽  
Author(s):  
SR Mudge ◽  
WR Lewis-Henderson ◽  
RG Birch
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Ccd Camera ◽  
Reporter Gene Expression ◽  
In Vitro Assays ◽  
E Coli ◽  
Cell Extracts ◽  
Cooled Ccd

Luciferase genes from Vibrio harveyi (luxAB) and firefly (luc) were introduced into E. coli, Agrobacteriurn, Arabidopsis and tobacco. Transformed bacteria and plants were quantitatively assayed for luciferase activity using a range of in vitro and in vivo assay conditions. Both lux and luc proved efficient reporter genes in bacteria, although it is important to be aware that the sensitive assays may detect expression due to readthrough from distant promoters. LUX activity was undetectable by liquid nitrogen-cooled CCD camera assays on intact tissues of plants which showed strong luxAB expression by in vitro assays. The decanal substrate for the lux assay was toxic to many plant tissues, and caused chemiluminescence in untransformed Arabidopsis leaves. These are serious limitations to application of the lux system for sensitive, non-toxic assays of reporter gene expression in plants. In contrast, LUC activity was readily detectable in intact tissues of all plants with luc expression detectable by luminometer assays on cell extracts. Image intensities of luc-expressing leaves were commonly two to four orders of magnitude above controls under the CCD camera. Provided adequate penetration of the substrate luciferin is obtained, luc is suitable for applications requiring sensitive, non-toxic assays of reporter gene expression in plants.

The relationship between digestibility or energy losses as methane and metabolisable energy content of whole crop wheat

1996 ◽  
Vol 1996 ◽  
pp. 41-41
Author(s):  
A.T. Adesogan ◽  
E. Owen ◽  
D.I. Givens
Keyword(s):  
Organic Matter ◽  
Energy Content ◽  
Organic Matter Content ◽  
Matter Content ◽  
Energy Losses ◽  
Digestible Organic Matter ◽  
Metabolisable Energy ◽  
The Cost ◽  
The Relationship

Estimates of the metabolisable energy (ME) content of whole crop wheat (WCW) derived using measured energy losses as methane (ELMm) are lacking due to the cost of measuring ELMm. Published ME values of WCW are largely calculated using predicted energy losses as methane (ELMp, Blaxter and Clapperton, 1965) or digestible organic matter content (DOMD) in vivo. However, there appears to be no published information about the accuracy with which DOMD in vivo or ELMp predicts the ME content of WCW. Therefore, this study assessed the validity of such ME predictions by comparing them with ME contents calculated using ELMm.

scholarly journals Role of F1C Fimbriae, Flagella, and Secreted Bacterial Components in the Inhibitory Effect of Probiotic Escherichia coli Nissle 1917 on Atypical Enteropathogenic E. coli Infection

2014 ◽  
Vol 82 (5) ◽  
pp. 1801-1812 ◽  
Author(s):  
Sylvia Kleta ◽  
Marcel Nordhoff ◽  
Karsten Tedin ◽  
Lothar H. Wieler ◽  
Rafal Kolenda ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Inhibitory Effect ◽  
Host Cells ◽  
Single Infection ◽  
Content Type ◽  
E Coli ◽  
Initial Adhesion

ABSTRACTEnteropathogenicEscherichia coli(EPEC) is recognized as an important intestinal pathogen that frequently causes acute and persistent diarrhea in humans and animals. The use of probiotic bacteria to prevent diarrhea is gaining increasing interest. The probioticE. colistrain Nissle 1917 (EcN) is known to be effective in the treatment of several gastrointestinal disorders. While bothin vitroandin vivostudies have described strong inhibitory effects of EcN on enteropathogenic bacteria, including pathogenicE. coli, the underlying molecular mechanisms remain largely unknown. In this study, we examined the inhibitory effect of EcN on infections of porcine intestinal epithelial cells with atypical enteropathogenicE. coli(aEPEC) with respect to single infection steps, including adhesion, microcolony formation, and the attaching and effacing phenotype. We show that EcN drastically reduced the infection efficiencies of aEPEC by inhibiting bacterial adhesion and growth of microcolonies, but not the attaching and effacing of adherent bacteria. The inhibitory effect correlated with EcN adhesion capacities and was predominantly mediated by F1C fimbriae, but also by H1 flagella, which served as bridges between EcN cells. Furthermore, EcN seemed to interfere with the initial adhesion of aEPEC to host cells by secretion of inhibitory components. These components do not appear to be specific to EcN, but we propose that the strong adhesion capacities enable EcN to secrete sufficient local concentrations of the inhibitory factors. The results of this study are consistent with a mode of action whereby EcN inhibits secretion of virulence-associated proteins of EPEC, but not their expression.

scholarly journals Identification of an Adeno-Associated Virus Rep Protein Binding Site in the Adenovirus E2a Promoter

2005 ◽  
Vol 79 (1) ◽  
pp. 28-38 ◽  
Author(s):  
John M. Casper ◽  
Jennifer M. Timpe ◽  
John David Dignam ◽  
James P. Trempe
Keyword(s):  
Gene Expression ◽  
Random Sequence ◽  
Gene Promoter ◽  
Helper Virus ◽  
Host Cells ◽  
Mobility Shift ◽  
Adeno Associated Virus ◽  
Rep Protein

ABSTRACT Adeno-associated virus (AAV) and other parvoviruses inhibit proliferation of nonpermissive cells. The mechanism of this inhibition is not thoroughly understood. To learn how AAV interacts with host cells, we investigated AAV's interaction with adenovirus (Ad), AAV's most efficient helper virus. Coinfection with Ad and AAV results in an AAV-mediated inhibition of Ad5 gene expression and replication. The AAV replication proteins (Rep) activate and repress gene expression from AAV and heterologous transcription promoters. To investigate the role of Rep proteins in the suppression of Ad propagation, we performed chromatin immunoprecipitation analyses that demonstrated in vivo AAV Rep protein interaction with the Ad E2a gene promoter. In vitro binding of purified AAV Rep68 protein to the Ad E2a promoter was characterized by electrophoretic mobility shift assays (Kd = 200 ± 25 nM). A 38 bp, Rep68-protected region (5′-TAAGAGTCAGCGCGCAGTATTTACTGAAGAGAGCCT-3′) was identified by DNase I footprint analysis. The 38-bp protected region contains the weak E2a TATA box, sequence elements that resemble the Rep binding sites identified by random sequence oligonucleotide selection, and the transcription start site. These results suggest that Rep binding to the E2a promoter contributes to the inhibition of E2a gene expression from the Ad E2a promoter and may affect Ad replication.

scholarly journals How the colonic environment influences Enterohaemorrhagic E. coli outer membrane vesicle production, and the interaction between outer membrane vesicles with human host cells

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Daniel Yara ◽  
Regis Stentz ◽  
Tom Wileman ◽  
Stephanie Schuller
Keyword(s):  
Outer Membrane ◽  
Bile Salts ◽  
Membrane Vesicle ◽  
Outer Membrane Proteins ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Host Cells ◽  
T84 Cells ◽  
E Coli

Enterohaemorrhagic E. coli (EHEC) may instigate bloody diarrhoea and haemolytic uraemic syndrome (HUS) due to Shiga toxin (Stx) production. Stx has been detected within outer membrane vesicles (OMVs), which are membrane-derived nanosized proteoliposomes. During colonisation, EHEC encounters many environmental surroundings such as the presence of bile salts and carbon dioxide (CO2). Here, the influence of different intestinal cues on EHEC OMV production was studied. OMV yield was quantified by densitometric analysis of outer membrane proteins F/C and A, following OMV protein separation by SDS-PAGE. Compared to cultures in Luria broth, higher OMV yields were attained following culture in human cell growth medium and simulated colonic environmental medium, with further increases in the presence of bile salts. Interestingly, lower yields were attained in the presence of T84 cells and CO2. The interaction between OMVs and different human cells was also examined by fluorescence microscopy. Here, OMVs incubated with cells showed internalisation by semi confluent but not fully confluent T84 cell monolayers. OMVs were internalised into the lysosomes in confluent Vero and Caco-2 cells, with Stx being transported to the Golgi and then the Endoplasmic reticulum. OMVs were detected within polarised Caco-2 cells, with no impact on the transepithelial electrical resistance by 24 hours. These results suggest that the colonic environmental factors influences OMV production in vivo. Additionally, results highlight the discrepancies which arise when using different cells lines to examine the intestine. Nevertheless, coupled with Stx, OMVs may serve as tools of EHEC which are involved in HUS development.

Sign in / Sign up

Export Citation Format

Share Document