Engineering of Therapeutic and Diagnostic O-Glycans on Recombinant Mucin-Type Immunoglobulin Fusion Proteins Expressed in CHO Cells

2013 ◽  
pp. 3-17 ◽  
Author(s):  
Linda Lindberg ◽  
Jining Liu ◽  
Jan Holgersson
Keyword(s):  
Cho Cells ◽  
Fusion Proteins

scholarly journals Evaluation of the Effects of Human Beta-Interferon Scaffold Attachment Region (IFN-SAR) on Expression of Vascular Endothelial Growth Factor-Fc (VEGF-Fc) Fusion Protein Expression in Chinese Hamster Ovary (CHO) Cells

2020 ◽  
Vol 26 (4) ◽  
pp. 393-398
Author(s):  
Ehsan Naghneh ◽  
Es'hagh Pourmaleki ◽  
Azam Rahimpour
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Monoclonal Antibodies ◽  
Fusion Protein ◽  
Cho Cells ◽  
Fusion Proteins ◽  
Chinese Hamster ◽  
Vascular Endothelial ◽  
Cell Clones ◽  
Fc Fusion Protein ◽  
Endothelial Growth Factor

Background: Recombinant anti-vascular endothelial growth factor (VEGF) monoclonal antibodies and Fc-fusion proteins have been widely used for the effective treatment of retinal neovascular diseases. In this regard, VEGFR-Fc fusions, which act as strong VEGF inhibitors, have been approved for the treatment of age-related macular degeneration (AMD) and diabetic macular edema (DME). Production of monoclonal antibodies and Fc-fusion proteins relies on mammalian host systems such as Chinese hamster ovary (CHO) cells. Application of genomic regulatory elements including scaffold/matrix attachment regions (SAR/MARs) can profoundly affect recombinant protein expression in CHO cells. Methods: To construct the VEGFR-Fc expression vectors, the enhanced green fluorescent protein (EGFP) gene was replaced by the VEGFR-Fc coding sequence in pEGFP-SAR-puro and pEGFP-puro vectors. Recombinant plasmids were transfected to CHO-K1 cells using TurboFect transfection reagent. VEGFR-Fc expression was evaluated in transiently transfected cells as well as stable cell pools and clones using an enzyme-linked immunosorbent assay (ELISA). Results: IFN-SAR showed no significant effect on transient expression of VEGFR-Fc during 72 h of culture. However, a 2.2-fold enhancement in VEGFR-Fc fusion protein titer was observed in IFN-SAR containing stable cell pools. Further evaluation of the VEGFR-Fc expression level in single-cell clones also indicated that clones with the highest VEGFR-Fc expression belonged to the pools transfected with IFN-SAR construct. Conclusion: Our results indicate that the incorporation of IFN-SAR in expression vector can increase the expression of VEGFR-Fc in stable cell pools as well as single-cell clones. In contrast, transient expression of the fusion protein was not affected by IFN-SAR. More studies are needed to investigate the mechanism underlying this effect, including the analysis of mRNA expression and gene copy number in stable cell pools as well as clonal cells.

Erythropoietin Fusion Proteins Comprised of Identical Head-to-Tail Repeats with Enhanced Biological Activity.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1162-1162
Author(s):  
Jee-Yeong Jeong ◽  
Changmin Chen ◽  
Kerry L. Davis ◽  
Andreas Breidbach ◽  
Don H. Catlin ◽  
...  
Keyword(s):  
Amino Acid ◽  
Fusion Protein ◽  
Cho Cells ◽  
Half Life ◽  
Fusion Proteins ◽  
Biological Activities ◽  
Cmv Promoter ◽  
Equivalent Unit

Abstract Recombinant human erythropoietin (EPO, epoetin) is used widely for treatment of chronic anemia due to renal failure, cancer, and other causes. However, considerably high and frequent doses of EPO are required to maintain therapeutic effectiveness, since it has a relatively short in vivo half-life. Thus, alternatives with higher efficacy and/or longer half-life are being developed. We have shown previously that EPO-dimers, either produced by chemical cross-linking of monomeric EPO or expressed as a recombinant fusion protein from COS cells, exhibit enhanced biological properties in vitro and in vivo (Sytkowski, et.al. Proc. Natl. Acad. Sci. USA 95, 1184; Sytkowski, et.al. J. Biol. Chem. 274, 24773). We now report increased activities of EPO-dimer fusion protein and EPO-trimer fusion protein comprised of identical head-to-tail repeats and a 15 or 20-amino acid linker (for dimer), or 17-amino acid linkers (for trimer) produced from stably transfected CHO cells. EPO-fusion proteins were expressed under a CMV promoter with a signal peptide present on the first monomer coding sequence. The EPO-dimer fusion protein was connected with either three or four repeats of Gly-Gly-Gly-Gly-Ser as a 15 or 20-amino acid linker sequence, respectively. The expression levels of EPO-dimer fusion protein from cloned CHO cells to supernatant of protein-free medium ranged from 4 to 40 mg/L determined by EPO-ELISA, and from 2.0×105 to 4.5×106 IU/L determined by in vitro bioassay. We selected clones producing EPO-dimer fusion protein with the greatest extent of glycosylation, as indicated by SDS-PAGE and isoelectric focusing. Subcutaneous injection of mice with three doses of EPO-dimer fusion protein resulted in percent increases in mean hematocrit of 32.6% (300 IU/kg) or 18.2% (100 IU/kg), while equivalent unit doses of EPO-monomer increased mean hematocrit by 12.5% (300 IU/kg) or 6.4% (100 IU/kg). Moreover, a single dose of EPO-dimer fusion protein (100 IU/kg) increased their mean hematocrit by 4.3% within 7 days, while an equivalent unit dose of EPO-monomer had no effect. Importantly, three doses of EPO-trimer fusion protein increased their mean hematocrit by 8.83% per IU injected, which was much greater than that observed with EPO-monomer (0.69%) or EPO-dimer fusion protein (1.81%). The results show that EPO-fusion proteins exhibit biological activities superior to those of EPO-monomer, suggesting important therapeutic advantages.

GPR31 and GPR151 are activated under acidic conditions

2019 ◽  
Vol 166 (4) ◽  
pp. 317-322 ◽  
Author(s):  
Misaki Mashiko ◽  
Aya Kurosawa ◽  
Yuki Tani ◽  
Takashi Tsuji ◽  
Shigeki Takeda
Keyword(s):  
G Protein ◽  
Cho Cells ◽  
Fusion Proteins ◽  
Narrow Range ◽  
G Protein Coupled Receptors ◽  
Cellular Assays ◽  
Gtpγs Binding ◽  
Acidic Conditions ◽  
G Protein Coupled

Abstract Recent studies have revealed that not only proton-sensing channels, but also one family of G protein-coupled receptors (GPCRs) comprising OGR1, GPR4, G2A and TDAG8 are responsible for the sensing of extracellular protons, or pH. Here, we report that two other GPCRs, GPR31 and GPR151, were also activated in acidic condition. Elevated pH of assay mixtures resulted in a remarkable increase in [35S]GTPγS binding by GPR31–Giα and GPR151–Giα fusion proteins in a narrow range between pH 6 and 5. Our reporter gene assays with CHO cells expressing recombinant GPR31 or GPR151 also showed that activation was maximal at pH ∼5.8. Although these results from in vitro and cellular assays revealed slightly different pH sensitivities, all of our results indicated that GPR31 and GPR151 sensed extracellular protons equally well as other proton-sensing GPCRs.

Expression and bioactivity of recombinant human serum albumin and dTMP fusion proteins in CHO cells

2016 ◽  
Vol 100 (17) ◽  
pp. 7565-7575 ◽  
Author(s):  
Yi Ru ◽  
Dejuan Zhi ◽  
Dingding Guo ◽  
Yong Wang ◽  
Yang Li ◽  
...  
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Cho Cells ◽  
Fusion Proteins ◽  
Recombinant Human Serum Albumin

Metabolic engineering of CHO cells to prepare glycoproteins

2018 ◽  
Vol 2 (3) ◽  
pp. 433-442 ◽  
Author(s):  
Qiong Wang ◽  
Michael J. Betenbaugh
Keyword(s):  
Metabolic Engineering ◽  
Cho Cells ◽  
Genetic Manipulation ◽  
Chinese Hamster ◽  
Post Translational Modification ◽  
Effector Functions ◽  
Recombinant Glycoproteins ◽  
Production Platform ◽  
Chemical Inhibitors ◽  
Amino Acid Mutations

As a complex and common post-translational modification, N-linked glycosylation affects a recombinant glycoprotein's biological activity and efficacy. For example, the α1,6-fucosylation significantly affects antibody-dependent cellular cytotoxicity and α2,6-sialylation is critical for antibody anti-inflammatory activity. Terminal sialylation is important for a glycoprotein's circulatory half-life. Chinese hamster ovary (CHO) cells are currently the predominant recombinant protein production platform, and, in this review, the characteristics of CHO glycosylation are summarized. Moreover, recent and current metabolic engineering strategies for tailoring glycoprotein fucosylation and sialylation in CHO cells, intensely investigated in the past decades, are described. One approach for reducing α1,6-fucosylation is through inhibiting fucosyltransferase (FUT8) expression by knockdown and knockout methods. Another approach to modulate fucosylation is through inhibition of multiple genes in the fucosylation biosynthesis pathway or through chemical inhibitors. To modulate antibody sialylation of the fragment crystallizable region, expressions of sialyltransferase and galactotransferase individually or together with amino acid mutations can affect antibody glycoforms and further influence antibody effector functions. The inhibition of sialidase expression and chemical supplementations are also effective and complementary approaches to improve the sialylation levels on recombinant glycoproteins. The engineering of CHO cells or protein sequence to control glycoforms to produce more homogenous glycans is an emerging topic. For modulating the glycosylation metabolic pathways, the interplay of multiple glyco-gene knockouts and knockins and the combination of multiple approaches, including genetic manipulation, protein engineering and chemical supplementation, are detailed in order to achieve specific glycan profiles on recombinant glycoproteins for superior biological function and effectiveness.

Identification of common and distinct epigenetic re-programming properties of Core-Binding Factor (CBF) Fusion Proteins

2016 ◽  
Vol 228 (03) ◽  
Author(s):  
J Loke ◽  
A Ptasinska ◽  
MR Imperato ◽  
SA Assi ◽  
P Cauchy ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Core Binding Factor ◽  
Binding Factor

Cell-specific regulation of IRS-1 gene expression: role of E box and C/EBP binding site in HepG2 cells and CHO cells

Diabetes ◽  
1997 ◽  
Vol 46 (3) ◽  
pp. 354-362 ◽  
Author(s):  
K. Matsuda ◽  
E. Araki ◽  
R. Yoshimura ◽  
K. Tsuruzoe ◽  
N. Furukawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Cho Cells ◽  
Hepg2 Cells ◽  
E Box ◽  
Specific Regulation
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