Escherichia coli
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PROVIDER: PRJNA141515 | ENA |
REPOSITORIES: ENA
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Project description:Strong production of recombinant proteins interfere with cellular processes in many ways. The extent of the bacterial stress response is determined by the specific properties of the recombinant protein, and by the rates of transcription and translation. The consideration of bacterial stress and starvation responses is of crucial importance for the successful establishment of an industrial large scale bioprocess. Stress genes can be used as marker genes in order to monitor the fitness of industrial bacterial hosts during fermentation processes. For this purpose, here in our study we have applied transcriptome analysis for the description of general and specific stress and starvation responses of Escherichia coli. Producing recombinant protein (Xylanase) in high cell density fed batch culture.
2011-05-24 | GSE29439 | GEO
Project description:Strong production of recombinant proteins interfere with cellular processes in many ways. The extent of the bacterial stress response is determined by the specific properties of the recombinant protein, and by the rates of transcription and translation. The consideration of bacterial stress and starvation responses is of crucial importance for the successful establishment of an industrial large scale bioprocess. Stress genes can be used as marker genes in order to monitor the fitness of industrial bacterial hosts during fermentation processes. For this purpose, here in our study we have applied transcriptome analysis for the description of general and specific stress and starvation responses of Escherichia coli. Producing recombinant protein (Xylanase) in high cell density fed batch culture. Cells were grown in batch mode in TB media till an OD600 of 15. The specific growth rate of 0.3 h-1 was maintained by fed batch started at OD600 of 15. Cells were induced at an OD600 of 30 using 1 mM IPTG and the growth and product profile was monitored. Samples at different time points (2hrs, 4hrs, and 6hrs) were taken and frozen in liquid nitrogen quickly for transcriptome analysis.
2011-05-23 | E-GEOD-29439 | biostudies-arrayexpress
Project description:Fed-batch cultivation of recombinant Chinese hamster ovary (CHO) cell lines is one of the most widely used production mode for commercial manufacturing of recombinant protein therapeutics. Furthermore, fed-batch cultivations are often conducted as biphasic processes where culture temperature is decreased to maximize volumetric product yields. However, it still remains to be elucidated which intracellular regulatory elements actually control the observed pro-productive phenotypes. Recently, several studies have revealed microRNAs (miRNAs) to be important molecular switches of cell phenotypes since single miRNAs are capable of regulating entire physiological pathways. In this study, we analyzed miRNA profiles of two different recombinant CHO cell lines (high and low producer), and compared them to a non-producing CHO DG44 host cell line during fed-batch cultivation at 37 versus 30 °C culture temperature. Taking advantage of next-generation sequencing combined with cluster, correlation and differential expression analyses, we could identify 89 different miRNAs, which might be interesting for CHO cell engineering. Functional validation experiments using 19 validated target miRNAs confirmed that these miRNAs indeed induced changes in process relevant phenotypes such as recombinant protein production, apoptosis, necrosis and proliferation. Furthermore, computational miRNA target prediction combined with functional clustering identified putative target genes and cellular pathways, which might be regulated by these miRNAs. Taken together, our study systematically identified novel target miRNAs during different phases and conditions of a biphasic fed-batch process and functionally evaluated their potential for host cell engineering.
2016-04-03 | GSE72945 | GEO
Project description:Fed-batch cultivation of recombinant Chinese hamster ovary (CHO) cell lines is one of the most widely used production mode for commercial manufacturing of recombinant protein therapeutics. Furthermore, fed-batch cultivations are often conducted as biphasic processes where culture temperature is decreased to maximize volumetric product yields. However, it still remains to be elucidated which intracellular regulatory elements actually control the observed pro-productive phenotypes. Recently, several studies have revealed microRNAs (miRNAs) to be important molecular switches of cell phenotypes since single miRNAs are capable of regulating entire physiological pathways. In this study, we analyzed miRNA profiles of two different recombinant CHO cell lines (high and low producer), and compared them to a non-producing CHO DG44 host cell line during fed-batch cultivation at 37 versus 30 °C culture temperature. Taking advantage of next-generation sequencing combined with cluster, correlation and differential expression analyses, we could identify 89 different miRNAs, which might be interesting for CHO cell engineering. Functional validation experiments using 19 validated target miRNAs confirmed that these miRNAs indeed induced changes in process relevant phenotypes such as recombinant protein production, apoptosis, necrosis and proliferation. Furthermore, computational miRNA target prediction combined with functional clustering identified putative target genes and cellular pathways, which might be regulated by these miRNAs. Taken together, our study systematically identified novel target miRNAs during different phases and conditions of a biphasic fed-batch process and functionally evaluated their potential for host cell engineering. 36 miRNA libraries from three different CHO cell lines and two process condition. In the control run temperature was maintained at 30°C, while temperature was reduced to 30°C after reaching mid exponential phase
2016-04-03 | E-GEOD-72945 | biostudies-arrayexpress
Project description:Chinese Hamster ovary (CHO) cells are the main platform used to produce recombinant proteins in the biopharmaceutical industry and are commonly cultured in either fed-batch or perfusion mode. However, the optimization of the complex biological systems used in such processes is extremely challenging. Omics approaches can reveal otherwise unknown characteristics of these systems and identify culture parameters that can be manipulated to optimize the cultivation process. Here we have applied proteomic profiling to a monoclonal antibody (mAb) production operated in perfusion mode to explore how cell biology and reactor environment change as the cell density reaches ≥ 200 x 106 cells/mL. The proteomics data show an increase of structural proteins as cell density increase, signs of oxidative stress and changes in glutathione metabolism at very high cell densities. Additionally, metabolomic profiling was carried out. See article “High cell density culture has a maintained exoproteome and metabolome” for more information.
2018-07-04 | PXD008760 | Pride
Project description:Over expression of recombinant proteins is known to cause a metabolic burden to the host cells which leads to down regulation of both growth rates and protein expression. Most studies in this regard have been conducted in low density shake flask cultures which does not capture the essential features of an industrial scale bioprocess. In the present work we studied the transcriptomic profiling at different specific growth rates while expressing the recombinant human interferon beta in fed batch cultures with complex media. These conditions mimicked the industrial fermentations for recombinant proteins. To determine the transcriptomic response of E.coli BL21(DE3) to recombinant protein production at three different specific growth rates, samples were collected for four time points (0h, 2h, 4h, 6h) post induction for the fed batch cultivations at specific growth rates 0.2h-1, 0.3h-1, 0.4h-1 respectively and induced at an optical density of 30. 0h (uninduced) sample was taken as control conditions for every run.
2011-04-06 | E-GEOD-28412 | biostudies-arrayexpress
Project description:The transcriptome profiles for wild-type (plasmid-free) and recombinant (plasmid-bearing) Escherichia coli during well-controlled synchronized high-cell-density fed-batch cultures were analyzed by DNA microarrays. It was observed that the growth phase significantly affected the transcriptome profiles, and the transcriptome profiles were significantly different for the recombinant and wild-type cultures. The response of the wild-type and recombinant cultures to an isopropyl-1-thio-β-D-galactopyranoside- (IPTG-) addition was examined, where IPTG induced recombinant protein production in the plasmid-bearing cultures. The IPTG-addition significantly altered the transcriptome response of the wild-type cultures entering the stationary phase. The IPTG-induced recombinant protein production resulted in a significant down-regulation of many energy synthesis genes (atp, nuo, cyo), as well as nearly all transcription- and translation-related genes (rpo, rpl, rpm, rps, rrf, rrl, rrs). Numerous phage (psp, hfl) and transposon-related genes (tra, ins) were significantly regulated in the recombinant cultures due to the IPTG-induction. These results indicate that the signaling mechanism, associated with the recombinant protein production, may induce a metabolic burden in the form of a phage defense mechanism. Taken together, these results indicated that recombinant protein production initiated a cascade of transcriptome responses that down-regulated the very genes needed to sustain productivity. In this study, the transcriptome profiles for recombinant and wild-type E. coli cultures were compared. The recombinant and wild-type cultures were monitored during the exponential growth phase and as the cultures entered the stationary phase. Well-controlled fed-batch fermenters were used to synchronize the high-cell density cultures. E. coli MG1655 [pPROEx-CAT] and E. coli MG1655 (plasmid-free) were exposed to IPTG in the exponential phase, where chloramphenicol acetyltransferase (CAT) expression was induced in the plasmid-bearing cultures. Control cultures, not exposed to IPTG, were also examined. The gene expression profiles were determined using DNA microarrays. Recombinant protein production was shown to result in a metabolic burden due to significant down-regulation of the transcription, translation, and energy synthesis genes. This was the first comprehensive study that has examined the transcriptome of wild-type and recombinant cultures under the same set of conditions. This data indicated that one cannot merely extrapolate the behavior of recombinant cultures from wild-type culture data.
2009-08-07 | GSE17505 | GEO
Project description:Over expression of recombinant proteins is known to cause a metabolic burden to the host cells which leads to down regulation of both growth rates and protein expression. Most studies in this regard have been conducted in low density shake flask cultures which does not capture the essential features of an industrial scale bioprocess. In the present work we studied the transcriptomic profiling at different specific growth rates while expressing the recombinant human interferon beta in fed batch cultures with complex media. These conditions mimicked the industrial fermentations for recombinant proteins.
2011-04-07 | GSE28412 | GEO
Project description:The transcriptome profiles for wild-type (plasmid-free) and recombinant (plasmid-bearing) Escherichia coli during well-controlled synchronized high-cell-density fed-batch cultures were analyzed by DNA microarrays. It was observed that the growth phase significantly affected the transcriptome profiles, and the transcriptome profiles were significantly different for the recombinant and wild-type cultures. The response of the wild-type and recombinant cultures to an isopropyl-1-thio-β-D-galactopyranoside- (IPTG-) addition was examined, where IPTG induced recombinant protein production in the plasmid-bearing cultures. The IPTG-addition significantly altered the transcriptome response of the wild-type cultures entering the stationary phase. The IPTG-induced recombinant protein production resulted in a significant down-regulation of many energy synthesis genes (atp, nuo, cyo), as well as nearly all transcription- and translation-related genes (rpo, rpl, rpm, rps, rrf, rrl, rrs). Numerous phage (psp, hfl) and transposon-related genes (tra, ins) were significantly regulated in the recombinant cultures due to the IPTG-induction. These results indicate that the signaling mechanism, associated with the recombinant protein production, may induce a metabolic burden in the form of a phage defense mechanism. Taken together, these results indicated that recombinant protein production initiated a cascade of transcriptome responses that down-regulated the very genes needed to sustain productivity. In this study, the transcriptome profiles for recombinant and wild-type E. coli cultures were compared. The recombinant and wild-type cultures were monitored during the exponential growth phase and as the cultures entered the stationary phase. Well-controlled fed-batch fermenters were used to synchronize the high-cell density cultures. E. coli MG1655 [pPROEx-CAT] and E. coli MG1655 (plasmid-free) were exposed to IPTG in the exponential phase, where chloramphenicol acetyltransferase (CAT) expression was induced in the plasmid-bearing cultures. Control cultures, not exposed to IPTG, were also examined. The gene expression profiles were determined using DNA microarrays. Recombinant protein production was shown to result in a metabolic burden due to significant down-regulation of the transcription, translation, and energy synthesis genes. This was the first comprehensive study that has examined the transcriptome of wild-type and recombinant cultures under the same set of conditions. This data indicated that one cannot merely extrapolate the behavior of recombinant cultures from wild-type culture data. Experiment Overall Design: All fermentations were synchronized in time to the time at which the cell density reached approximately 11.5 OD, or roughly 70% of the fermenter’s maximum cell density. IPTG (5 mM) was added when the cell density reached the targeted OD for the cultures to be induced. Cells were harvested at 0, 1, 2, 3, 4, and 5 hours relative to the synchronization time point (identified as Time S0, S1, etc). Each fermentation condition was conducted in duplicate. For the recombinant Control Time S0 sample, triplicate samples were available since prior to the IPTG-addition, all fermentations within a strain were replicates. All fermentation conditions were repeated twice (two biological replicates). RNA from each biological replicate was purified and processed independently. Three biological replicates were obtained from for the recombinant Control sample (Time S0) since the cultures prior to the IPTG-addition were replicates. Prior to hybridization, where only two biological replicates existed, one of the RNA samples was divided into two technical replicates, resulting in three separate hybridized chips. The wild-type Time S0 samples and all Time S1 and S4 samples contained data from three DNA microarrays obtained from two biological duplicates (30 DNA microarrays total).
2009-08-25 | E-GEOD-17505 | biostudies-arrayexpress
Project description:We analyzed the transcriptomic response in three distinct CHO cell lines: a non-producing host cell line (CHOZN®GS-/-), an immunoglobulin G1 (IgG1) producer, and an erythropoietin Fc fusion (EPO-Fc) producer. We compared the growth and production characteristics of all three cell lines during fed-batch culture. High throughput RNA sequencing (RNASeq) and quantitative polymerase chain reaction (qPCR) were used to study differential gene expression analysis of the timecourse dataset with the host cell line CHOZN®GS-/- as the reference. The objective of this analysis was to study the common and unique responses of cell lines producing different protein products. This analysis showed enrichment and transient upregulation of mRNAs involved in ER stress, the UPR, and oxidative protein folding and processing in both protein producers. Analysis of temporal differential expression profiles led to the identification of novel engineering targets.
2024-06-26 | GSE217637 | GEO