High-level synthesis of recombinant murine endostatin in Chinese hamster ovary cells

Chura-Chambi, Rosa Maria; Tornieri, P.H; Spencer, Patrick Jack; Nascimento, P.A.; Mathor, Mônica Beatriz; Morganti, Lígia

doi:10.1016/j.pep.2004.01.003

Cited by 8 publications

(4 citation statements)

References 17 publications

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“…Both rmEs and his 6 -mEs inhibited endothelial cell proliferation, in a dose dependent manner. 57 In spite of these advantages, this system has some disadvantages including: high cost, laborious production, time-consuming, poor secretion and also potential for contamination of the product by viruses. 58 …”

Section: Clinical Application Challengesmentioning

confidence: 99%

The Challenges of Recombinant Endostatin in Clinical Application: Focus on the Different Expression Systems and Molecular Bioengineering

Mohajeri¹,

Sanaei

Kiafar³

et al. 2017

Adv Pharm Bull

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Angiogenesis plays an essential role in rapid growing and metastasis of the tumors. Inhibition of angiogenesis is a putative strategy for cancer therapy. Endostatin (Es) is an attractive anti-angiogenesis protein with some clinical application challenges including; short half-life, instability in serum and requirement to high dosage. Therefore, production of recombinant endostatin (rEs) is necessary in large scale. The production of rEs is difficult because of its structural properties and is high-cost. Therefore, this review focused on the different expression systems that involved in rEs production including; mammalian, baculovirus, yeast, and Escherichia coli (E. coli) expression systems. The evaluating of the results of different expression systems declared that none of the mentioned systems can be considered to be generally superior to the other. Meanwhile with considering the advantages and disadvantage of E. coli expression system compared with other systems beside the molecular properties of Es, E. coli expression system can be a preferred expression system for expressing of the Es in large scale. Also, the molecular bioengineering and sustained release formulations that lead to improving of its stability and bioactivity will be discussed. Point mutation (P125A) of Es, addition of RGD moiety or an additional zinc biding site to N-terminal of Es , fusing of Es to anti-HER2 IgG or heavy-chain of IgG, and finally loading of the endostar by PLGA and PEG- PLGA nanoparticles and gold nano-shell particles are the effective bioengineering methods to overcome to clinical changes of endostatin.

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Section: Clinical Application Challengesmentioning

confidence: 99%

The Challenges of Recombinant Endostatin in Clinical Application: Focus on the Different Expression Systems and Molecular Bioengineering

Mohajeri¹,

Sanaei

Kiafar³

et al. 2017

Adv Pharm Bull

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“…The GCR cDNA was cloned into the pED dicistronic expression vector, which is commonly used to obtain high-expression levels of heterologous proteins in mammalian cells [45–47], and positive clones were isolated and amplified with increasing MTX concentration up to 700 nM MTX. Probably due to the inability of the transfected cells to further amplify the dhfr gene [28], higher concentration of MTX (1200 nM) caused cell death.…”

Section: Discussionmentioning

confidence: 99%

“…Although the highest productivity achieved for secreted GCR was calculated as 5.14 pg/cell/day, higher expression levels have been reported for other recombinant proteins in CHO cells [45, 47]. Here, it should be taken into account the fact that GCR is a membrane-associated glycoprotein secreted at low level or rather not secreted under natural conditions [37].…”

Section: Discussionmentioning

confidence: 99%

Generation of a Chinese Hamster Ovary Cell Line Producing Recombinant Human Glucocerebrosidase

Novo

Morganti

Moro

et al. 2012

Journal of Biomedicine and Biotechnology

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Impaired activity of the lysosomal enzyme glucocerebrosidase (GCR) results in the inherited metabolic disorder known as Gaucher disease. Current treatment consists of enzyme replacement therapy by administration of exogenous GCR. Although effective, it is exceptionally expensive, and patients worldwide have a limited access to this medicine. In Brazil, the public healthcare system provides the drug free of charge for all Gaucher's patients, which reaches the order of $ 84 million per year. However, the production of GCR by public institutions in Brazil would reduce significantly the therapy costs. Here, we describe a robust protocol for the generation of a cell line producing recombinant human GCR. The protein was expressed in CHO-DXB11 (dhfr−) cells after stable transfection and gene amplification with methotrexate. As expected, glycosylated GCR was detected by immunoblotting assay both as cell-associated (~64 and 59 kDa) and secreted (63–69 kDa) form. Analysis of subclones allowed the selection of stable CHO cells producing a secreted functional enzyme, with a calculated productivity of 5.14 pg/cell/day for the highest producer. Although being laborious, traditional methods of screening high-producing recombinant cells may represent a valuable alternative to generate expensive biopharmaceuticals in countries with limited resources.

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“…Chinese hamster ovary (CHO) cells transformed with the vector pED- endo, expressing 28.8 μg of (his) 6 -met-endostatin (ES)/10 6 cells/24 h (5) were maintained in α-minimum essential medium (α-MEM; Gibco, USA) supplemented with 50 U/ml penicilin, 50 μg/ml streptomycin, 1.25 μg/ml amphotericin B, 2 mM glutamine, and 10% heat-inactivated fetal bovine serum. C-PAE cells (ATCC CCL-209) were maintained in Eagle's minimal essential medium (EMEM) supplemented with 20% FBS, 50 U/ml penicillin, 50 μg/ml streptomycin, and 2 mM l-glutamine.…”

Section: Methodsmentioning

confidence: 99%

Continuous and High-Level in Vivo Delivery of Endostatin from Recombinant Cells Encapsulated in TheraCyte® Immunoisolation Devices

et al. 2010

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Endostatin (ES) is a potent inhibitor of angiogenesis and tumor growth. Continuous ES delivery of ES improves the efficacy and potency of the antitumoral therapy. The TheraCyte system is a polytetrafluoroethylene (PTFE) semipermeable membrane macroencapsulation system for implantation of genetically engineered cells specially designed for the in vivo delivery of therapeutic proteins, such as ES, which circumvents the problem of limited half-life and variation in circulating levels. In order to enable neovascularization at the tissues adjacent to the devices prior to ES secretion by the cells inside them, we designed a scheme in which empty TheraCyte devices were preimplanted SC into immunodeficient mice. Only after healing (17 days later) were Chinese hamster ovary cells expressing ES injected into the preimplanted devices. In another model for device implantation, the cells expressing ES where loaded into the immunoisolation devices prior to implantation into the animals, and the TheraCyte were then immediately implanted SC into the mice. Throughout the 2-month study, constant high ES levels of up to 3.7 microg/ml were detected in the plasma of the mice preimplanted with the devices, while lower but also constant levels of ES (up to 2.1 microg/ml plasma) were detected in the mice that had received devices preloaded with the ES-expressing cells. Immunohistochemistry using anti-ES antibody showed reaction within the device and outside it, demonstrating that ES, secreted by the confined recombinant cells, permeated through the membrane and reached the surrounding tissues.

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High-level synthesis of recombinant murine endostatin in Chinese hamster ovary cells

Cited by 8 publications

References 17 publications

The Challenges of Recombinant Endostatin in Clinical Application: Focus on the Different Expression Systems and Molecular Bioengineering

The Challenges of Recombinant Endostatin in Clinical Application: Focus on the Different Expression Systems and Molecular Bioengineering

Generation of a Chinese Hamster Ovary Cell Line Producing Recombinant Human Glucocerebrosidase

Continuous and High-Level in Vivo Delivery of Endostatin from Recombinant Cells Encapsulated in TheraCyte® Immunoisolation Devices

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