Enhancement of human erythropoietin production in Chinese hamster ovary cells through supplementation of 30Kc19-30Kc6 fusion protein

et al. 2016

Biotechnology Journal

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The protein‐stabilizing and cell‐penetrating activities of Bombyx mori 30Kc19 α‐helix domain (30Kc19α) are investigated. Recently, 30Kc19 protein has been studied extensively as it has both protein‐stabilizing and cell‐penetrating properties. However, it is unknown which part of 30Kc19 is responsible for those properties. 30Kc19 protein is composed of two distinct domains, an α‐helix N‐terminal domain (30Kc19α) and a β‐trefoil C‐terminal domain (30Kc19β). The authors construct and produce truncated forms of 30Kc19 to demonstrate their biological functions. Interestingly, 30Kc19α was shown to be responsible for both the protein‐stabilizing and cell‐penetrating properties of 30Kc19 protein. 30Kc19α shows even higher protein delivery activity than did whole 30Kc19 protein and has low cytotoxicity when added to cell culture medium. Therefore, based on its multifunctional properties, 30Kc19α can be developed as a novel candidate for a therapeutic protein carrier into various cells and tissues.

Section: Introductionmentioning

confidence: 99%

Protein‐stabilizing and cell‐penetrating properties of α‐helix domain of 30Kc19 protein

Ryu

Kim

et al. 2016

Biotechnology Journal

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“…In this study, a CHO cell line was obtained and used to produce recombinant human EPO (EPO-CHO), as described in previous studies [23,30]. Briefly, exponentially growing cells were seeded into dishes and cultivated for three days in DMEM/F-12 medium supplemented with 10% fetal bovine serum (FBS, Welgene, Daejeon, Korea), 100 U/mL penicillin, and 100 µg/mL streptomycin (complete medium) at 37 • C in a 5% CO 2 -humidified incubator (BB15, Thermo Fisher Scientific).…”

Section: Cell Culturementioning

confidence: 99%

Recombinant Human Erythropoietin Production in Chinese Hamster Ovary Cells Is Enhanced by Supplementation of α-Helix Domain of 30Kc19 Protein

Cha

2021

Applied Sciences

The enhancement of recombinant therapeutic protein production in mammalian cell culture has been regarded as an important issue in the biopharmaceutical industry. Previous studies have reported that the addition of the recombinant 30Kc19 protein, a silkworm-derived plasma protein with simultaneous cell-penetrating and mitochondrial enzyme-stabilizing properties, can enhance the recombinant protein expression in Chinese hamster ovary (CHO) cell culture. Here, we produced an α-helix N-terminal domain of 30Kc19, called (30Kc19α), and investigated its effects on the production of human erythropoietin (EPO), a widely used therapeutic protein for the treatment of anemia, in recombinant CHO cell culture. Similar to the full-length 30Kc19, 30Kc19α was able to be mass-produced in a form of recombinant protein through an Escherichia coli expression system and delivered into EPO-producing CHO (EPO–CHO) cells. Supplementing the medium of EPO–CHO cell culture with 30Kc19α increased the intracellular NADPH/NADP+ ratio related to the flux of metabolic reducing power for protein biosynthesis, subsequently enhancing EPO production in serum-free culture. 30Kc19α is considered to have certain advantages in the downstream purification process of therapeutic protein production when it is used as a medium supplement due to its small size and low isoelectric point compared to the full-length 30Kc19. These results suggest that 30Kc19α has potential use for manufacturing biopharmaceutical proteins.

“…30Kc19 protein is a member of the 30K protein family found in silkworm hemolymph, having molecular weights around 30 kDa. In our previous studies, anti-apoptotic effect was observed in various cells via 30K gene expression or addition of 30K proteins in recombinant form produced from Escherichia coli (Choi et al 2006;Choi et al 2002;Choi et al 2005;Ha and Park 1997;Ha et al 1996;Kim et al 2004;Park et al 2015;Rhee et al 1999;Rhee et al 2002;Rhee and Park 2000;Wang et al 2011). Moreover, in recent studies, cell-penetrating and enzyme-stabilizing effects of 30Kc19 protein were also observed (Park et al 2014;Park et al 2012a;Park et al 2012b).…”

Section: Introductionmentioning

confidence: 99%

α-Galactosidase delivery using 30Kc19-human serum albumin nanoparticles for effective treatment of Fabry disease

Lee

Appl Microbiol Biotechnol

Sohn

et al. 2016

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Fabry disease is a genetic lysosomal storage disease caused by deficiency of α-galactosidase, the enzyme-degrading neutral glycosphingolipid that is transported to lysosome. Glycosphingolipid accumulation by this disease causes multi-organ dysfunction and premature death of the patient. Currently, enzyme replacement therapy (ERT) using recombinant α-galactosidase is the only treatment available for Fabry disease. To maximize the efficacy of treatment, enhancement of cellular delivery and enzyme stability is a challenge in ERT using α-galactosidase. In this study, protein nanoparticles using human serum albumin (HSA) and 30Kc19 protein, originating from silkworm, were used to enhance the delivery and intracellular α-galactosidase stability. 30Kc19-HSA nanoparticles loaded with the α-galactosidase were formed by desolvation method. 30Kc19-HSA nanoparticles had a uniform spherical shape and were well dispersed in cell culture media. 30Kc19-HSA nanoparticles had negligible toxicity to human cells. The nanoparticles exhibited enhanced cellular uptake and intracellular stability of delivered α-galactosidase in human foreskin fibroblast. Additionally, they showed enhanced globotriaosylceramide degradation in Fabry patients' fibroblasts. It is expected that 30Kc19-HSA protein nanoparticles could be used as an effective tool for efficient delivery and enhanced stability of drugs.