scholarly journals Engineering mammalian cells to produce plant-specific N-glycosylation on proteins

Glycobiology ◽  
2020 ◽  
Vol 30 (8) ◽  
pp. 528-538 ◽  
Author(s):  
Joachim Steen Larsen ◽  
Richard Torbjörn Gustav Karlsson ◽  
Weihua Tian ◽  
Morten Alder Schulz ◽  
Annemarie Matthes ◽  
...  
Keyword(s):  
Cho Cells ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Structural Features ◽  
Host Cells ◽  
Igg Antibody ◽  
Endogenous Genes ◽  
Species Specific ◽  
Full Conversion ◽  
Core Fucosylation

Abstract Protein N-glycosylation is an essential and highly conserved posttranslational modification found in all eukaryotic cells. Yeast, plants and mammalian cells, however, produce N-glycans with distinct structural features. These species-specific features not only pose challenges in selecting host cells for production of recombinant therapeutics for human medical use but also provide opportunities to explore and utilize species-specific glycosylation in design of vaccines. Here, we used reverse cross-species engineering to stably introduce plant core α3fucose (α3Fuc) and β2xylose (β2Xyl) N-glycosylation epitopes in the mammalian Chinese hamster ovary (CHO) cell line. We used directed knockin of plant core fucosylation and xylosylation genes (AtFucTA/AtFucTB and AtXylT) and targeted knockout of endogenous genes for core fucosylation (fut8) and elongation (B4galt1), for establishing CHO cells with plant N-glycosylation capacities. The engineering was evaluated through coexpression of two human therapeutic N-glycoproteins, erythropoietin (EPO) and an immunoglobulin G (IgG) antibody. Full conversion to the plant-type α3Fuc/β2Xyl bi-antennary agalactosylated N-glycosylation (G0FX) was demonstrated for the IgG1 produced in CHO cells. These results demonstrate that N-glycosylation in mammalian cells is amenable for extensive cross-kingdom engineering and that engineered CHO cells may be used to produce glycoproteins with plant glycosylation.

scholarly journals PERKEMBANGAN TEKNOLOGI SEL MAMALIA CHINESE HAMSTER OVARY (CHO) UNTUK PRODUKSI OBAT BERBASIS PROTEIN

2019 ◽  
Vol 18 (2) ◽  
pp. 125-133
Author(s):  
Adi Santoso
Keyword(s):  
Cho Cells ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Basic Research ◽  
Host Cells ◽  
Basic Knowledge ◽  
Research Progress ◽  
Market Demand ◽  
Technological Advances ◽  
Development Technology

Chinese hamsters ovary (CHO) and its derivative such as CHO-DXB11 cells, CHO-K1, CHO-DG44 and CHO-S are mammalian cells that are often used for production of therapeutic protein drugs. The CHO cells often used for protein production have several advantages including 1) host cells that are safe to use in drug production, 2) the level of production of proteins produced can be increased by amplifying genes using methotrexate (MTX), 3) having the capacity to make post-translation modificationsand 4) CHO cells can be adapted to grow in suspension. The high need for protein-based drugs triggers the development of basic knowledge and innovation in production of recombinant proteins. The impressive technological advances in CHO cell technology have made these cells can be used to produce proteins around 10 g/liter in order to meet the market demand. The first protein successfully produced using CHO mammalian cells was the therapeutic Tissue Plasminogen Activator (r-tPA, Activase) protein used for stroke patients. The presence of this drug is quickly followed by several other types of drugs. In this review, history of development of CHO cells, the contribution of CHO cells to basic research, progress of effective line cell screening and development technology are discussed.

scholarly journals Structural and functional characterization of SARS-CoV-2 RBD domains produced in mammalian cells

2021 ◽  
Author(s):  
Christoph Gstöttner ◽  
Tao Zhang ◽  
Anja Resemann ◽  
Sophia Ruben ◽  
Stuart Pengelley ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Mammalian Cells ◽  
Vaccine Development ◽  
Functional Characterization ◽  
Chinese Hamster ◽  
Structural Features ◽  
Host Cells ◽  
Hek293 Cells ◽  
Functional Features

AbstractAs the SARS-CoV-2 pandemic is still ongoing and dramatically influences our life, the need for recombinant proteins for diagnostics, vaccine development, and research is very high. The spike (S) protein, and particularly its receptor binding domain (RBD), mediates the interaction with the ACE2 receptor on host cells and may be modulated by its structural features. Therefore, well characterized recombinant RBDs are essential. We have performed an in-depth structural and functional characterization of RBDs expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK293) cells. To structurally characterize the native RBDs (comprising N- and O-glycans and additional posttranslational modifications) a multilevel mass spectrometric approach was employed. Released glycan and glycopeptide analysis were integrated with intact mass analysis, glycan-enzymatic dissection and top-down sequencing for comprehensive annotation of RBD proteoforms. The data showed distinct glycosylation for CHO- and HEK293-RBD with the latter exhibiting antenna fucosylation, higher level of sialylation and a combination of core 1 and core 2 type O-glycans. Additionally, from both putative O-glycosylation sites, we could confirm that O-glycosylation was exclusively present at T323, which was previously unknown. For both RBDs, the binding to SARS-CoV-2 antibodies of positive patients and affinity to ACE2 receptor was addressed showing comparable results. This work not only offers insights into RBD structural and functional features but also provides a workflow for characterization of new RBDs and batch-to-batch comparison.

scholarly journals Neurotrophin Receptor TrkC Is an Entry Receptor for Trypanosoma cruzi in Neural, Glial, and Epithelial Cells

2011 ◽  
Vol 79 (10) ◽  
pp. 4081-4087 ◽  
Author(s):  
Craig Weinkauf ◽  
Ryan Salvador ◽  
Mercio PereiraPerrin
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Neuronal Cell ◽  
Host Cells ◽  
Neurotrophin Receptor ◽  
Content Type

ABSTRACTTrypanosoma cruzi, the agent of Chagas' disease, infects a variety of mammalian cells in a process that includes multiple cycles of intracellular division and differentiation starting with host receptor recognition by a parasite ligand(s). Earlier work in our laboratory showed that the neurotrophin-3 (NT-3) receptor TrkC is activated byT. cruzisurfacetrans-sialidase, also known as parasite-derived neurotrophic factor (PDNF). However, it has remained unclear whether TrkC is used byT. cruzito enter host cells. Here, we show that a neuronal cell line (PC12-NNR5) relatively resistant toT. cruzibecame highly susceptible to infection when overexpressing human TrkC but not human TrkB. Furthermore,trkCtransfection conferred an ∼3.0-fold intracellular growth advantage. Sialylation-deficient Chinese hamster ovarian (CHO) epithelial cell lines Lec1 and Lec2 also became much more permissive toT. cruziafter transfection with thetrkCgene. Additionally, NT-3 specifically blockedT. cruziinfection of the TrkC-NNR5 transfectants and of naturally permissive TrkC-bearing Schwann cells and astrocytes, as did recombinant PDNF. Two specific inhibitors of Trk autophosphorylation (K252a and AG879) and inhibitors of Trk-induced MAPK/Erk (U0126) and Akt kinase (LY294002) signaling, but not an inhibitor of insulin-like growth factor 1 receptor, abrogated TrkC-mediated cell invasion. Antibody to TrkC blockedT. cruziinfection of the TrkC-NNR5 transfectants and of cells that naturally express TrkC. The TrkC antibody also significantly and specifically reduced cutaneous infection in a mouse model of acute Chagas' disease. TrkC is ubiquitously expressed in the peripheral and central nervous systems, and in nonneural cells infected byT. cruzi, including cardiac and gastrointestinal muscle cells. Thus, TrkC is implicated as a functional PDNF receptor in cell entry, independently of sialic acid recognition, mediating broadT. cruziinfection bothin vitroandin vivo.

scholarly journals Establishment of fast-growing serum-free immortalised cells from Chinese hamster lung tissues for biopharmaceutical production

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Noriko Yamano-Adachi ◽  
Rintaro Arishima ◽  
Sukwattananipaat Puriwat ◽  
Takeshi Omasa
Keyword(s):  
Cell Line ◽  
Cho Cells ◽  
Mammalian Cells ◽  
High Efficiency ◽  
Chinese Hamster ◽  
Serum Free ◽  
Sterility Testing ◽  
Fast Growing ◽  
Defined Culture

Abstract Chinese hamster (Cricetulus griseus) ovary-derived Chinese hamster ovary (CHO) cells are the most commonly used mammalian hosts for the industrial production of recombinant therapeutics because of their ability to fold, assemble, and perform post-translational modifications, such as glycosylation, on proteins. They are also valuable for their ability to grow in serum-free suspension cultures. In this study, we established a cell line derived from lung tissue of Chinese hamsters, named Chinese hamster lung (CHL)-YN cells. The biosafety of CHL-YN cells was confirmed by in vitro sterility testing, mycoplasma detection, and reverse transcriptase assays. One of the key characteristics of CHL-YN cells was their doubling time of 8.1 h in chemically defined culture medium; thus, they proliferate much faster than conventional CHO cells and general mammalian cells. Transgenes could be introduced into CHL-YN cells with high efficiency. Finally, between 50% to > 100% of the amount of glycosylated immunoglobulin G (IgG)1 produced by CHO-K1 cells was produced by CHL-YN cells over a shorter period of time. In summary, fast-growing CHL-YN cells are a unique cell line for producing recombinant proteins.

scholarly journals Heparan Sulfate Mediates Infection of High-Neurovirulence Theiler's Viruses

2002 ◽  
Vol 76 (16) ◽  
pp. 8400-8407 ◽  
Author(s):  
Honey V. Reddi ◽  
Howard L. Lipton
Keyword(s):  
Heparan Sulfate ◽  
Mammalian Cells ◽  
Virus Entry ◽  
Chinese Hamster ◽  
Host Cells ◽  
Direct Access ◽  
Neuraminidase Treatment ◽  
Encephalomyelitis Virus ◽  
Protein Entry ◽  
Theiler's Viruses

ABSTRACT The mechanisms by which Theiler's murine encephalomyelitis virus (TMEV) binds and enters host cells and the molecules involved are not completely understood. In this study, we demonstrate that the high-neurovirulence TMEV GDVII virus uses the glycosaminoglycan heparan sulfate (HS) as an attachment factor that is required for efficient infection. Studies based on soluble HS-mediated inhibition of attachment and infection, removal of HS with specific enzymes, and blocking with anti-HS antibodies establish that HS mediates GDVII virus entry into mammalian cells. Data from defined proteoglycan-deficient Chinese hamster ovary mutant cells further support the role of HS in GDVII infection and indicate that the extent of sulfation is critical for infection. Neuraminidase treatment of proteoglycan-deficient cells restores permissiveness to GDVII virus, indicating that sialic acid hinders direct access of virus to the protein entry receptor. A model of the potential steps in GDVII virus entry into mammalian cells involving HS is proposed.

scholarly journals A Drosophila ribosomal protein functions in mammalian cells.

1990 ◽  
Vol 10 (9) ◽  
pp. 4524-4528 ◽  
Author(s):  
C G Maki ◽  
D D Rhoads ◽  
J J Diaz ◽  
D J Roufa
Keyword(s):  
Ribosomal Protein ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Expression Vector ◽  
Resistance Mutation ◽  
Chinese Hamster ◽  
Ribosomal Subunits ◽  
Cdna Expression ◽  
Protein Functions

A cDNA expression vector encoding Drosophila ribosomal protein S14 was transfected into cultured Chinese hamster ovary (CHO) cells that harbor a recessive RPS14 emetine resistance mutation. Transformants synthesized the insect mRNA and polypeptide and consequently displayed an emetine-sensitive phenotype. These observations indicate that the insect protein was accurately expressed and correctly assembled into functional mammalian 40S ribosomal subunits.

scholarly journals Interaction of Ipa proteins of Shigella flexneri with alpha5beta1 integrin promotes entry of the bacteria into mammalian cells.

1996 ◽  
Vol 183 (3) ◽  
pp. 991-999 ◽  
Author(s):  
M Watarai ◽  
S Funato ◽  
C Sasakawa
Keyword(s):  
Epithelial Cells ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Shigella Flexneri ◽  
Chinese Hamster ◽  
Invasive Capacity ◽  
Beta 1 Integrin ◽  
Cellular Components

Shigella is a genus of highly adapted bacterial pathogens that cause bacillary dysentery in humans. Bacteria reaching the colon invade intestinal epithelial cells by a process of bacterial-directed endocytosis mediated by the Ipa proteins: IpaB, IpaC, and IpaD of Shigella. The invasion of epithelial cells is thought to be a receptor-mediated phenomenon, although the cellular components of the host that interact with the Ipa proteins have not yet been identified. We report here that in a Shigella flexneri invasive system and Chinese hamster ovary (CHO) cell monolayers, the Ipa proteins were capable of interacting directly with alpha5beta1 integrin. The invasive capacity of S. flexneri for CHO cells increased as levels of alpha5beta1 integrin were elevated. When CHO cells were infected with S. flexneri, the tyrosine phosphorylation both of pp 125FAK, an integrin-regulated 125 K focal adhesion kinase, and of paxillin was stimulated. In contrast, an isogenic strain of S. flexneri that was defective in invasion owing to a mutation in its spa32 gene failed to induce such phosphorylation. Under in vitro and in vivo conditions, the released IpaB, IpaC, and IpaD proteins bound to alpha 5 beta 1 integrin in a manner different from that of soluble fibronectin but similar to that of the tissue form of fibronectin. At the site of attachment of S. flexneri to CHO cells, alpha5beta1 integrin converged with polymerization of actin. These data thus suggest that the capacity of Ipa proteins to interact with alpha5beta1 integrin may be an important Shigella factor in triggering the reorganization of actin cytoskeletons.

scholarly journals Engineering of Chinese Hamster Ovary Cells With NDPK-A to Enhance DNA Nuclear Delivery Combined With EBNA1 Plasmid Maintenance Gives Improved Exogenous Transient Reporter, mAb and SARS-CoV-2 Spike Protein Expression

2021 ◽  
Vol 9 ◽  
Author(s):  
James D. Budge ◽  
Robert J. Young ◽  
Christopher Mark Smales
Keyword(s):  
Recombinant Protein ◽  
Plasmid Dna ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Transient Gene Expression ◽  
Chinese Hamster ◽  
Nuclear Antigen ◽  
Stable Cell Line ◽  
Nuclear Pore

Transient gene expression (TGE) in mammalian cells is a method of rapidly generating recombinant protein material for initial characterisation studies that does not require time-consuming processes associated with stable cell line construction. High TGE yields are heavily dependent on efficient delivery of plasmid DNA across both the plasma and nuclear membranes. Here, we harness the protein nucleoside diphosphate kinase (NDPK-A) that contains a nuclear localisation signal (NLS) to enhance DNA delivery into the nucleus of CHO cells. We show that co-expression of NDPK-A during transient expression results in improved transfection efficiency in CHO cells, presumably due to enhanced transportation of plasmid DNA into the nucleus via the nuclear pore complex. Furthermore, introduction of the Epstein Barr Nuclear Antigen-1 (EBNA-1), a protein that is capable of inducing extrachromosomal maintenance, when coupled with complementary oriP elements on a transient plasmid, was utilised to reduce the effect of plasmid dilution. Whilst there was attenuated growth upon introduction of the EBNA-1 system into CHO cells, when both NDPK-A nuclear import and EBNA-1 mediated technologies were employed together this resulted in enhanced transient recombinant protein yields superior to those generated using either approach independently, including when expressing the complex SARS-CoV-2 spike (S) glycoprotein.

A Drosophila ribosomal protein functions in mammalian cells

1990 ◽  
Vol 10 (9) ◽  
pp. 4524-4528
Author(s):  
C G Maki ◽  
D D Rhoads ◽  
J J Diaz ◽  
D J Roufa
Keyword(s):  
Ribosomal Protein ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Expression Vector ◽  
Resistance Mutation ◽  
Chinese Hamster ◽  
Ribosomal Subunits ◽  
Cdna Expression ◽  
Protein Functions

A cDNA expression vector encoding Drosophila ribosomal protein S14 was transfected into cultured Chinese hamster ovary (CHO) cells that harbor a recessive RPS14 emetine resistance mutation. Transformants synthesized the insect mRNA and polypeptide and consequently displayed an emetine-sensitive phenotype. These observations indicate that the insect protein was accurately expressed and correctly assembled into functional mammalian 40S ribosomal subunits.

The Use of Allele-Specific Recombinant Fcγ Receptor IIIb Antigens for the Detection of Granulocyte Antibodies

Blood ◽  
1999 ◽  
Vol 93 (1) ◽  
pp. 357-362 ◽  
Author(s):  
Juergen Bux ◽  
Karin Kissel ◽  
Christine Hofmann ◽  
Sentot Santoso
Keyword(s):  
Flow Cytometry ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Fcγ Receptor ◽  
High Background ◽  
Mammalian Expression ◽  
Human Sera ◽  
Allele Specific ◽  
Granulocyte Antibodies

The Fcγ receptor IIIb (FcγRIIIb) for the Fc domain of IgG is expressed exclusively on neutrophils. The FcγRIIIb bears allotypic polymorphisms referred to as NA1, NA2, and SH, which are known for their frequent involvement in alloimmune and autoimmune neutropenias as well as in transfusion reactions. The bactericidal capacity of isolated neutrophils is easily activatable, and activation results in self-desintegration, thus preventing storage of neutrophils. As a result, only freshly isolated granulocytes can be used for antibody screening, often making it impossible to use typed panel cells. To provide a readily available source of typed panel cells, we therefore established stable mammalian cells expressing recombinant NA1, NA2, and SH antigens. We isolated mRNA from typed neutrophils and then transcribed it in cDNA. The cDNA that codes for the different forms of the FcγRIIIb was amplified by polymerase chain reaction and was subsequently subcloned into the mammalian expression vector pcDNA3. Chinese hamster ovary (CHO) cells were transfected with allele-specific constructs, and stable cell lines expressing FcγRIIIb were selected by flow cytometry. Because human sera show high background fluorescence with transfectants in flow cytometry, the monoclonal antibody–specific isolation of granulocyte antigens (MAIGA) assay was performed. By MAIGA assay, we tested 14 well-characterized human alloantibodies directed against the antigens NA1, NA2, and SH; 5 FcγRIIIb-specific isoantibodies; and 12 FcγRIIIb-reactive autoantibodies. Except one NA1- and one SH-specific alloantibody, all other antibodies could be identified by the use of CHO transfectants. In contrast to neutrophils, fixed CHO cells can be stored at 4°C for at least 4 weeks or stored frozen for a longer period. This longer shelf life of the transfected CHO cells compared with isolated neutrophils will simplify the detection of the clinically most important FcγRIIIb-reactive alloantibodies and autoantibodies.

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