Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Rescue of the complex temperature-sensitive phenotype of Chinese hamster ovary E36ts20 cells by expression of the human ubiquitin-activating enzyme cDNA.

ABSTRACT: The ubiquitin conjugation system is a multi-step pathway in which ubiquitin is activated and conjugated to acceptor proteins, one function of which is to target acceptor proteins for rapid degradation within the cell. The conjugation system is involved in many aspects of cellular functions, including the cell cycle. Several cell-cycle arrest mutant cell lines have been characterized and appear to harbour a mutant ubiquitin-activating enzyme, E1, as their primary defect. One such cell line is ts20, which is derived from Chinese hamster ovary E36 cells. This cell line has been used to characterize some of the potential functions of the ubiquitin conjugation system in vivo, such as its involvement in the maturation of autophagic vacuoles. The present study describes the complete rescue of the complex ts20 phenotype following the expression of the cDNA for human E1. Stable transfectants expressing the human E1 cDNA in the CMVneo expression vector were measured for ubiquitin-conjugation activity, protein degradation and growth in culture at the nonpermissive temperature. This rescue confirms that the phenotype observed in the ts20 cells is due to a defect in the E1 enzyme. Thus, the ts20 cell line will serve as a useful tool to delineate the functions of the ubiquitin system in vivo.

SUBMITTER: Handley-Gearhart PM

PROVIDER: S-EPMC1137433 | biostudies-other | 1994 Dec

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

GeneOptimizer program-assisted cDNA reengineering enhances sRAGE autologous expression in Chinese hamster ovary cells.

Project description:Soluble receptor for advanced glycation end products (sRAGE) is a secreted mammalian protein that functions as a decoy to counter-react RAGE signaling-resultant pathological conditions, and has high therapeutic potentials. Our prior studies showed that recombinant human sRAGE expressed in Chinese hamster, Ceanothus griseus, ovary (CHO) cells is modified by specific N-glycosylation, and exhibits higher bioactivity than that expressed in other host systems including insect Spodoptera frugiperda cells. Here, we show that GeneOptimizer software program-assisted, reengineered sRAGE cDNA enhances the recombinant protein expression in CHO cells. The cDNA sequence encoding human sRAGE was optimized for RNA structure, stability, and codon usages in CHO cells. We found that such optimization augmented sRAGE expression over 2 folds of its wild-type counterpart. We also studied how individual parameter impacted sRAGE autologous expression in CHO cells, and whether sRAGE bioactivity was compromised. We found that the enhanced expression appeared not to affect sRAGE N-glycosylation and bioactivity. Optimization of sRAGE expression provides a basis for future large-scale production of this protein to meet medical needs.

| S-EPMC3936886 | biostudies-literature

A metabolic CRISPR-Cas9 screen in Chinese hamster ovary cells identifies glutamine-sensitive genes.

Project description:Media and feed optimization have fueled many-fold improvements in mammalian biopharmaceutical production, but genome editing offers an emerging avenue for further enhancing cell metabolism and bioproduction. However, the complexity of metabolism, involving thousands of genes, makes it unclear which engineering strategies will result in desired traits. Here we present a comprehensive pooled CRISPR screen for CHO cell metabolism, including ~16,000 gRNAs against ~2500 metabolic enzymes and regulators. Using this screen, we identified a glutamine response network in CHO cells. Glutamine is particularly important since it is often over-fed to drive increased TCA cycle flux, but toxic ammonia may accumulate. With the screen we found one orphan glutamine-responsive gene with no clear connection to our network. Knockout of this novel and poorly characterized lipase, Abhd11, substantially increased growth in glutamine-free media by altering the regulation of the TCA cycle. Thus, the screen provides an invaluable targeted platform to comprehensively study genes involved in any metabolic trait, and elucidate novel regulators of metabolism.

| S-EPMC8193919 | biostudies-literature

Proteomic analysis of Chinese hamster ovary cells.

Project description:To complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO cells including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis, multidimensional liquid chromatography, and solid phase extraction of glycopeptides (SPEG). From the 120 different mass spectrometry analyses generating 682,097 MS/MS spectra, 93,548 unique peptide sequences were identified with at most 0.02 false discovery rate (FDR). A total of 6164 grouped proteins were identified from both glycoproteome and proteome analysis, representing an 8-fold increase in the number of proteins currently identified in the CHO proteome. Furthermore, this is the first proteomic study done using the CHO genome exclusively, which provides for more accurate identification of proteins. From this analysis, the CHO codon frequency was determined and found to be distinct from humans, which will facilitate expression of human proteins in CHO cells. Analysis of the combined proteomic and mRNA data sets indicated the enrichment of a number of pathways including protein processing and apoptosis but depletion of proteins involved in steroid hormone and glycosphingolipid metabolism. Five-hundred four of the detected proteins included N-acetylation modifications, and 1292 different proteins were observed to be N-glycosylated. This first large-scale proteomic analysis will enhance the knowledge base about CHO capabilities for recombinant expression and provide information useful in cell engineering efforts aimed at modifying CHO cellular functions.

| S-EPMC3772721 | biostudies-literature

Mitotic spindle proteomics in Chinese hamster ovary cells.

Project description:Mitosis is a fundamental process in the development of all organisms. The mitotic spindle guides the cell through mitosis as it mediates the segregation of chromosomes, the orientation of the cleavage furrow, and the progression of cell division. Birth defects and tissue-specific cancers often result from abnormalities in mitotic events. Here, we report a proteomic study of the mitotic spindle from Chinese Hamster Ovary (CHO) cells. Four different isolations of metaphase spindles were subjected to Multi-dimensional Protein Identification Technology (MudPIT) analysis and tandem mass spectrometry. We identified 1155 proteins and used Gene Ontology (GO) analysis to categorize proteins into cellular component groups. We then compared our data to the previously published CHO midbody proteome and identified proteins that are unique to the CHO spindle. Our data represent the first mitotic spindle proteome in CHO cells, which augments the list of mitotic spindle components from mammalian cells.

| S-EPMC3103581 | biostudies-literature

Comprehensive Glycoproteomic Analysis of Chinese Hamster Ovary Cells.

Project description:The Chinese hamster ovary (CHO) cell line is a major expression system for the production of therapeutic proteins, the majority of which are glycoproteins, such as antibodies and erythropoietin (EPO). The characterization glycosylation profile of therapeutic proteins produced from engineered CHO cells and therapeutic functions, as well as side effects, are critical to understand the important roles of glycosylation. In this study, a large scale glycoproteomic workflow was established and applied to CHO-K1 cells expressing EPO. The workflow includes enrichment of intact glycopeptides from CHO-K1 cell lysate and medium using hydrophilic enrichment, fractionation of the obtained intact glycopeptides (IGPs) by basic reversed phase liquid chromatography (bRPLC), analyzing the glycopeptides using LC-MS/MS, and annotating the results by GPQuest 2.0. A total of 10 338 N-linked glycosite-containing IGPs were identified, representing 1162 unique glycosites in 530 glycoproteins, including 71 unique atypical N-linked IGPs on 18 atypical N-glycosylation sequons with an overrepresentation of the N-X-C motifs. Moreover, we compared the glycoproteins from CHO cell lysate with those from medium using the in-depth N-linked glycoproteome data. The obtained large scale glycoproteomic data from intact N-linked glycopeptides in this study is complementary to the genomic, proteomic, and N-linked glycomic data previously reported for CHO cells. Our method has the potential to monitor the production of recombinant therapeutic glycoproteins.

| S-EPMC6440468 | biostudies-literature

¹H NMR spectroscopy profiling of metabolic reprogramming of Chinese hamster ovary cells upon a temperature shift during culture.

Project description:We report an NMR based approach to determine the metabolic reprogramming of Chinese hamster ovary cells upon a temperature shift during culture by investigating the extracellular cell culture media and intracellular metabolome of CHOK1 and CHO-S cells during culture and in response to cold-shock and subsequent recovery from hypothermic culturing. A total of 24 components were identified for CHOK1 and 29 components identified for CHO-S cell systems including the observation that CHO-S media contains 5.6 times the level of glucose of CHOK1 media at time zero. We confirm that an NMR metabolic approach provides quantitative analysis of components such as glucose and alanine with both cell lines responding in a similar manner and comparable to previously reported data. However, analysis of lactate confirms a differentiation between CHOK1 and CHO-S and that reprogramming of metabolism in response to temperature was cell line specific. The significance of our results is presented using principal component analysis (PCA) that confirms changes in metabolite profile in response to temperature and recovery. Ultimately, our approach demonstrates the capability of NMR providing real-time analysis to detect reprogramming of metabolism upon cellular perception of cold-shock/sub-physiological temperatures. This has the potential to allow manipulation of metabolites in culture supernatant to improve growth or productivity.

| S-EPMC3795012 | biostudies-literature

Transient light-activated gene expression in Chinese hamster ovary cells.

Project description:BackgroundChinese hamster ovary (CHO) cells are widely used for industrial production of biopharmaceuticals. Many genetic, chemical, and environmental approaches have been developed to modulate cellular pathways to improve titers. However, these methods are often irreversible or have off-target effects. Development of techniques which are precise, tunable, and reversible will facilitate temporal regulation of target pathways to maximize titers. In this study, we investigate the use of optogenetics in CHO cells. The light-activated CRISPR-dCas9 effector (LACE) system was first transiently transfected to express eGFP in a light-inducible manner. Then, a stable system was tested using lentiviral transduction.ResultsTransient transfections resulted in increasing eGFP expression as a function of LED intensity, and activation for 48 h yielded up to 4-fold increased eGFP expression compared to cells kept in the dark. Fluorescence decreased once the LACE system was deactivated, and a protein half-life of 14.9 h was calculated, which is in agreement with values reported in the literature. In cells stably expressing the LACE system, eGFP expression was confirmed, but there was no significant increase in expression following light activation.ConclusionsTaken together, these results suggest that optogenetics can regulate CHO cell cultures, but development of stable cell lines requires optimized expression levels of the LACE components to maintain high dynamic range.

| S-EPMC7863527 | biostudies-literature

Protein overproduction alters exosome secretion in Chinese hamster ovary cells.

Project description:Despite the abundance of available cell lines, nearly 70% of all recombinant therapeutic proteins today are produced in Chinese hamster ovary (CHO) cells. The impact of protein overproduction on the secretion of exosomes by CHO cells has been investigated here. Increased secretion of extracellular vesicles (EVs) by protein overexpressing CHO cells was demonstrated with protein content assay, nanoparticle tracking analysis, and capillary electrophoresis. Our results revealed that a protein overproduction might induce EVs secretion, which might be accompanied by the sequestration and loading of overexpressed proteins into the exosomes. These findings are of vital importance for the manufacturing of therapeutics in CHO expression systems due to the risk of product loss during downstream processing of culture medium as well as the application of exosomes as nanocarriers of therapeutic proteins. The study indicates also the importance of culturing process control.

| S-EPMC10287810 | biostudies-literature

Bioinformatic analysis of Chinese hamster ovary host cell protein lipases.

Project description:Complete, accurate genome assemblies are necessary to design targets for genetic engineering strategies. Successful gene knockdowns and knockouts in Chinese hamster ovary (CHO) cells may prevent the expression of difficult-to-remove host cell proteins (HCPs). HCPs, if not removed, can cause problems in stability, safety, and efficacy of the biotherapeutic. A significantly improved Chinese hamster (CH) reference genome was used to identify new knockout targets with similar predicted functions and characteristics as the difficult-to-remove host cell lipases, LPL, PLBL2, and LPLA2. The CHO-K1 gene and protein sequences of several of these lipases were corrected using the updated CH genome. Sequence alignments were then used to identify conserved regions that may serve as possible targets for multiple simultaneous gene knockouts. Finally, comparison of the CHO-K1 lipase protein sequences to their human orthologs provided insight into which lipases, if persistent in the drug product, could possibly cause immunogenic responses in patients.

| S-EPMC6430115 | biostudies-literature

Characterization of ubiquitin-activating enzyme Uba1 in the nucleus by its mammalian temperature-sensitive mutant.

Project description:Temperature-sensitive (ts) CHO-K1 mutant tsTM3 exhibits chromosomal instability and cell-cycle arrest in the S to G2 phases with decreased DNA synthesis at the nonpermissive temperature, 39°C. Previously, complementation tests with other mutants showed that tsTM3 harbors a genetic defect in the ubiquitin-activating enzyme Uba1. Sequence comparison of the Uba1 gene between wild-type and mutant cells in this study revealed that the mutant phenotype is caused by a G-to-A transition that yields a Met-to-Ile substitution at position 256 in hamster Uba1. The ts defects in tsTM3 were complemented by expression of the wild-type Uba1 tagged with green fluorescent protein. Expression of the Uba1 primarily in the nucleus appeared to rescue tsTM3 cells. Incubation at 39°C resulted in a decrease of nuclear Uba1 in tsTM3 cells, suggesting that loss of Uba1 in the nucleus may lead to the ts defects. Analyses with the fluorescent ubiquitination-based cell cycle indicator revealed that loss of function of Uba1 leads to failure of the ubiquitin system in the nucleus. Incubation at 39°C caused an increase in endogenous geminin in tsTM3 cells. A ts mutation of Uba1 found in tsTM3 cells appears to be a novel mutation reflecting the important roles of Uba1 in nucleus.

| S-EPMC4013028 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data