scholarly journals Comprehensive Glycoproteomic Analysis of Chinese Hamster Ovary Cells

2018 ◽  
Author(s):  
Ganglong Yang ◽  
Yingwei Hu ◽  
Shisheng Sun ◽  
Chuanzi Ouyang ◽  
Weiming Yang ◽  
...  
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Large Scale ◽  
Chinese Hamster Ovary Cells ◽  
Expression System ◽  
Chinese Hamster ◽  
Reversed Phase ◽  
Cho Cell ◽  
Cell Lysate ◽  
Therapeutic Glycoproteins

AbstractThe 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 of the glycosylation profiles is critical to understand the important role of glycosylation on therapeutic glycoproteins from CHO cells. 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 1,162 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 accelerate the production of recombinant therapeutic glycoproteins.

scholarly journals Cloning and characterization of small circular DNA from Chinese hamster ovary cells.

1984 ◽  
Vol 4 (1) ◽  
pp. 173-180 ◽  
Author(s):  
S W Stanfield ◽  
D R Helinski
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Single Copy ◽  
Chromosomal Dna ◽  
Cho Cell ◽  
Ovary Cells ◽  
Chromosomal Sequences

Small polydisperse circular (spc) DNA was isolated and cloned, using BglII from Chinese hamster ovary (CHO) cells. The properties of 47 clones containing at least 43 different BglII fragments are reported. The majority of the clones probably contain entire sequences from individual spcDNA molecules. Most of the clones were homologous to sequences in CHO cell chromosomal DNA, and many were also homologous to mouse LMTK- cell chromosomal sequences. The majority of homologous CHO cell chromosomal sequences were repetitive, although a few may be single copy. Only a small fraction of cloned spcDNA molecules were present in every cell; most occurred less frequently than once in 15 cells. Localization studies indicated that at least a portion of spcDNA is associated with the nucleus in CHO cells.

scholarly journals Evidence for intracellular partitioning of serine and glycine metabolism in Chinese hamster ovary cells

1996 ◽  
Vol 313 (3) ◽  
pp. 991-996 ◽  
Author(s):  
Michael R. NARKEWICZ ◽  
S. David SAULS ◽  
Susan S. TJOA ◽  
Cecilia TENG ◽  
Paul V. FENNESSEY
Keyword(s):  
Cell Lines ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Mitochondrial Protein ◽  
Chinese Hamster ◽  
Serine Hydroxymethyltransferase ◽  
Cho Cell ◽  
Ovary Cells ◽  
Glycine Metabolism

Serine hydroxymethyltransferase (SHMT) is the primary enzyme in the interconversion of serine and glycine. The roles of mitochondrial and cytosolic SHMT in the interconversion of serine and glycine were determined in two Chinese hamster ovary (CHO) cell lines that both contain cytosolic SHMT but either have (CHOm+) or lack (CHOm-) mitochondrial SHMT. Mitochondrial SHMT activity was significantly reduced in CHOm- (0.24±0.11 nmol/min per mg of mitochondrial protein) compared with CHOm+ (3.21±0.66 nmol/min per mg of mitochondrial protein; P = 0.02) cells, whereas cytosolic SHMT activity was similar in CHOm- and CHOm+ cells (1.09±0.31 and 1.53±0.12 nmol/min per mg of cytosolic protein respectively; P = 0.57). In CHOm+ and CHOm- cells, the relative flux of glycine to serine measured with either [1-13C]- or [2-13C]-glycine was similar (CHOm-: 538±82 nmol/24 per mg of DNA; CHOm+: 616±88 nmol/24 h per mg of DNA; P = 0.42). In contrast, the relative flux of serine to glycine measured with [1-13C]serine was low in CHOm- cells (80±28 nmol/24 h per mg of DNA) compared with CHOm+ cells (3080±320 nmol/24 h per mg of DNA; P = 0.0001). The rate of glycine production determined by UA-2[1-13C]glycine dilution was lower in CHOm- (1200±200 nmol/24 h per mg of DNA) than CHOm+ (10200±1800 nmol/24 h per mg of DNA; P = 0.03) cells, whereas glycine utilization was similar in the two cell lines. Serine production was similar in the two cell lines but serine utilization was lower in CHOm- (3800±1200 μmol/24 h per mg of DNA) than CHOm+ (6600±1000 nmol/24 h per mg of DNA; P = 0.0002) cells. Increasing the serine concentration in the medium resulted in an increase in glycine production in CHOm+ but not in CHOm- cells. Intracellular studies with [1-13C]serine confirm the findings of decreased glycine production from serine. In CHO cells there is partitioning of intracellular serine and glycine metabolism. Our data support the hypothesis that mitochondrial SHMT is the primary pathway for serine into glycine interconversion.

scholarly journals Differential involvement of cell surface sialic acid residues in wheat germ agglutinin binding to parental and wheat germ agglutinin-resistant Chinese hamster ovary cells.

1980 ◽  
Vol 85 (1) ◽  
pp. 60-69 ◽  
Author(s):  
P Stanley ◽  
T Sudo ◽  
J P Carver
Keyword(s):  
Sialic Acid ◽  
Cell Surface ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cho Cell ◽  
Differential Involvement

Two Chinese hamster ovary (CHO) cell mutants selected for resistance to wheat germ agglutinin (WGA) have been shown to exhibit defective sialylation of membrane glycoproteins and a membrane glycolipid, GM3. The mutants (termed WgaRII and WgaRIII) have been previously shown to belong to different genetic complementation groups and to exhibit different WGA-binding abilities. These mutants and a WGA-resistant CHO cell mutant termed WgaRI (which also possesses a surface sialylation defect arising from a deficient N-acetylglucosaminyltransferase activity), have enabled us to investigate the role of sialic acid in WGA binding at the cell surface. Scatchard plots of the binding of 125I-WGA (1 ng/ml to 1 mg/ml) to parental and WgaR CHO cells before and after a brief treatment with neuraminidase provide evidence for several different groups of sialic acid residues at the CHO cell surface which may be distinquished by their differential involvement in WGA binding to CHO cells.

scholarly journals CHOmpact: a reduced metabolic model of Chinese hamster ovary cells with enhanced interpretability

2021 ◽  
Author(s):  
Ioscani Jimenez del Val ◽  
Sarantos Kyriakopoulos ◽  
Simone Albrecht ◽  
Henning Stöckmann ◽  
Pauline M Rudd ◽  
...  
Keyword(s):  
Cell Culture ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Large Scale ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Reaction Network ◽  
Metabolic Model ◽  
Feeding Strategies ◽  
Metabolic Models

Metabolic modelling has emerged as a key tool for the characterisation of biopharmaceutical cell culture processes. Metabolic models have also been instrumental in identifying genetic engineering targets and developing feeding strategies that optimise the growth and productivity of Chinese hamster ovary (CHO) cells. Despite their success, metabolic models of CHO cells still present considerable challenges. Genome scale metabolic models (GeMs) of CHO cells are very large (>6000 reactions) and are, therefore, difficult to constrain to yield physiologically consistent flux distributions. The large scale of GeMs also makes interpretation of their outputs difficult. To address these challenges, we have developed CHOmpact, a reduced metabolic network that encompasses 101 metabolites linked through 144 reactions. Our compact reaction network allows us to deploy multi-objective optimisation and ensure that the computed flux distributions are physiologically consistent. Furthermore, our CHOmpact model delivers enhanced interpretability of simulation results and has allowed us to identify the mechanisms governing shifts in the anaplerotic consumption of asparagine and glutamate as well as an important mechanism of ammonia detoxification within mitochondria. CHOmpact, thus, addresses key challenges of large-scale metabolic models and, with further development, will serve as a platform to develop dynamic metabolic models for the control and optimisation of biopharmaceutical cell culture processes.

scholarly journals Controlling Ratios of Plasmid-Based Double Cut Donor and CRISPR/Cas9 Components to Enhance Targeted Integration of Transgenes in Chinese Hamster Ovary Cells

2021 ◽  
Vol 22 (5) ◽  
pp. 2407
Author(s):  
Sung Wook Shin ◽  
Dongwoo Kim ◽  
Jae Seong Lee
Keyword(s):  
Cell Line ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cho Cell ◽  
Cell Line Development ◽  
Cho Cell Line ◽  
Vector Systems ◽  
Targeted Integration

Chinese hamster ovary (CHO) cells are the most valuable expression host for the commercial production of biotherapeutics. Recent trends in recombinant CHO cell-line development have focused on the site-specific integration of transgenes encoding recombinant proteins over random integration. However, the low efficiency of homology-directed repair upon transfection of Cas9, single-guide RNA (sgRNA), and the donor template has limited its feasibility. Previously, we demonstrated that a double-cut donor (DCD) system enables highly efficient CRISPR/Cas9-mediated targeted integration (TI) in CHO cells. Here, we describe several CRISPR/Cas9 vector systems based on DCD templates using a promoter trap-based TI monitoring cell line. Among them, a multi-component (MC) system consisting of an sgRNA/DCD vector and Cas9 expression vector showed an approximate 1.5-fold increase in knock-in (KI) efficiency compared to the previous DCD system, when a systematically optimized relative ratio of sgRNA/DCD and Cas9 vector was applied. Our optimization efforts revealed that concurrently increasing sgRNA and DCD components relative to Cas9 correlated positively with KI efficiency at a single KI site. Furthermore, we explored component bottlenecks, such as effects of sgRNA components and applicability of the MC system on simultaneous double KI. Taken together, we improved the DCD vector design by tailoring plasmid constructs and relative component ratios, and this system can be widely used in the TI strategy of transgenes, particularly in CHO cell line development and engineering.

Cloning and characterization of small circular DNA from Chinese hamster ovary cells

1984 ◽  
Vol 4 (1) ◽  
pp. 173-180
Author(s):  
S W Stanfield ◽  
D R Helinski
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Single Copy ◽  
Chromosomal Dna ◽  
Cho Cell ◽  
Ovary Cells ◽  
Chromosomal Sequences

Small polydisperse circular (spc) DNA was isolated and cloned, using BglII from Chinese hamster ovary (CHO) cells. The properties of 47 clones containing at least 43 different BglII fragments are reported. The majority of the clones probably contain entire sequences from individual spcDNA molecules. Most of the clones were homologous to sequences in CHO cell chromosomal DNA, and many were also homologous to mouse LMTK- cell chromosomal sequences. The majority of homologous CHO cell chromosomal sequences were repetitive, although a few may be single copy. Only a small fraction of cloned spcDNA molecules were present in every cell; most occurred less frequently than once in 15 cells. Localization studies indicated that at least a portion of spcDNA is associated with the nucleus in CHO cells.

Review of the current methods of Chinese Hamster Ovary (CHO) cells cultivation for production of therapeutic protein

2020 ◽  
Vol 17 ◽  
Author(s):  
Shazid Md. Sharker ◽  
Md. Atiqur Rahman
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Mass Production ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Therapeutic Protein ◽  
Specific Productivity ◽  
Ovary Cells ◽  
Further Development ◽  
Interesting Area

Most of clinical approved protein-based drugs or under in clinical trial have a profound impact in the treatment of critical diseases. The mammalian eukaryotic cells culture approaches, particularly the CHO (Chinese Hamster Ovary) cells are mainly used in the biopharmaceutical industry for the mass-production of therapeutic protein. Recent advances in CHO cell bioprocessing to yield recombinant proteins and monoclonal antibodies have enabled the expression of quality protein. The developments of cell lines are possible to upgrade specific productivity. As a result, it holds an interesting area for academic as well as industrial researchers around the world. This review will concentrate on the recent progress of the mammalian CHO cells culture technology and the future scope of further development for the mass-production of protein therapeutics.

Expression of human Mn SOD in Chinese hamster ovary cells confers protection from oxidant injury

1993 ◽  
Vol 264 (6) ◽  
pp. L598-L605
Author(s):  
B. Warner ◽  
R. Papes ◽  
M. Heile ◽  
D. Spitz ◽  
J. Wispe
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Lethal Dose ◽  
Chinese Hamster ◽  
Eukaryotic Expression ◽  
Wild Type ◽  
Oxidant Injury ◽  
Sod Activity ◽  
Recombinant Cho Cells

Manganese superoxide dismutase (Mn SOD) is an important component of antioxidant defense in aerobic cells because of its location in the mitochondria, a significant source of oxygen radicals and an important target of oxidant injury. To test the hypothesis that increased mitochondrial Mn SOD protects from oxidant injury, Chinese hamster ovary (CHO) cells were transfected with a eukaryotic expression vector containing the human Mn SOD cDNA. In recombinant CHO cells, Mn SOD activity was increased threefold over wild-type controls. Acute survival during paraquat exposure (0–500 microM) was significantly improved in CHO cells expressing human Mn SOD, with 71% of recombinant CHO cells surviving at the 50% lethal dose (LD50) for wild-type CHO controls. Cell growth following exposure to paraquat (100 microM) was also significantly improved in recombinant CHO cells. CHO cells expressing human Mn SOD continued to grow and divide after paraquat exposure, whereas growth of wild-type CHO cells was negligible. Protection against oxidant-induced injury was directly related to increased Mn SOD, occurring in the absence of changes in other antioxidant enzymes including catalase, Cu,Zn SOD, and glutathione associated cellular antioxidant mechanisms. We conclude that increased expression of human Mn SOD in vitro directly confers protection against oxidant injury.

scholarly journals Continued initiation of DNA synthesis in arginine-deprived Chinese hamster ovary cells.

1977 ◽  
Vol 73 (1) ◽  
pp. 200-205 ◽  
Author(s):  
A S Weissfeld ◽  
H Rouse
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Multiplication ◽  
Ovary Cells ◽  
Cell Cycle Phases

When exponentially growing CHO cells were deprived of arginine (Arg), cell multiplication ceased after 12 h, but initiation of DNA synthesis continued: after 48 h of starvation with continuous [3H]thymidine exposure, 85% of the population had incorporated label, as detected autoradiographically. Consideration of the distribution of exponential cells in the various cell cycle phases leads to a calculation that most cells in G1 at the time that Arg was removed, as well as those in S, engaged in some DNA synthesis during starvation. In contrast, isoleucine (Ile)-starved cells did not initiate DNA synthesis, as has been reported by others. Experiments with cells synchronized by mitotic selection confirmed this difference in Arg- and Ile- deprived behavior, but also showed that cells which underwent the mitosis leads to G1 transition during Arg starvation remained arrested in G1 (G0?). The results suggest that Arg-deprived cells continue to maintain some proliferative function(s) while Ile-deprived cells do not.

scholarly journals Kinetics of endosome acidification in mutant and wild-type Chinese hamster ovary cells.

1987 ◽  
Vol 105 (6) ◽  
pp. 2713-2721 ◽  
Author(s):  
D J Yamashiro ◽  
F R Maxfield
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Ph Value ◽  
Ph Regulation ◽  
Chinese Hamster Ovary Cells ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Wild Type ◽  
Cell Biol ◽  
Endocytic Compartments

Acidification of endocytic compartments is necessary for the proper sorting and processing of many ligands and their receptors. Robbins and co-workers have obtained Chinese hamster ovary (CHO) cell mutants that are pleiotropically defective in endocytosis and deficient in ATP-dependent acidification of endosomes isolated by density centrifugation (Robbins, A. R., S. S. Peng, and J. L. Marshall. 1983. J. Cell Biol. 96:1064-1071; Robbins, A. R., C. Oliver, J. L. Bateman, S. S. Krag, C. J. Galloway, and I. Mellman. 1984. J. Cell Biol. 99:1296-1308). In this and the following paper (Yamashiro, D. J., and F. R. Maxfield. 1987. J. Cell Biol. 105:2723-2733) we describe detailed studies of endosome acidification in the mutant and wild-type CHO cells. Here we describe a new microspectrofluorometry method based on changes in fluorescein fluorescence when all cellular compartments are equilibrated to the same pH value. Using this method we measured the pH of endocytic compartments during the first minutes of endocytosis. We found in wild-type CHO cells that after 3 min, fluorescein-labeled dextran (F-Dex) was in endosomes having an average pH of 6.3. By 10 min, both F-Dex and fluorescein-labeled alpha 2-macroglobulin (F-alpha 2M) had reached acidic endosomes having an average pH of 6.0 or below. In contrast, endosome acidification in the CHO mutants DTG 1-5-4 and DTF 1-5-1 was markedly slowed. The average endosomal pH after 5 min was 6.7 in both mutant cell lines. At least 15 min was required for F-Dex and F-alpha 2M to reach an average pH of 6.0 in DTG 1-5-4. Acidification of early endocytic compartments is defective in the CHO mutants DTG 1-5-4 and DTF 1-5-1, but pH regulation of later compartments on both the recycling pathway and lysosomal pathway is nearly normal. The properties of the mutant cells suggest that proper functioning of pH regulatory mechanisms in early endocytic compartments is critical for many pH-mediated processes of endocytosis.

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