Expression and analysis of the glycosylation properties of recombinant human erythropoietin expressed in Pichia pastoris

Teh, Ser Huy; Fong, Mun Yik; Zeriouh, Mohamed

doi:10.1590/s1415-47572011005000022

Cited by 17 publications

(3 citation statements)

References 31 publications

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“…In case of rTbCLP, great variation can be seen in protein subpopulations. Similar results were previously described by Teh et al (39), where human erythropoietin (EPO) expressed in the P. pastoris system also demonstrated broad smear band in Western blot. EPO is a native glycoprotein (40), as is TbCLP and family 3 cystatins.…”

Section: Discussionsupporting

confidence: 90%

The Immunological Properties of Recombinant Multi-Cystatin-Like Domain Protein From Trichinella Britovi Produced in Yeast

et al. 2019

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Trichinellosis is a globally-distributed zoonotic parasitic disease caused by nematode worms of the genus Trichinella. One of the most common species of Trichinella known to affect human health is T. britovi; however, it is relatively poorly investigated. A thorough knowledge of the proteins expressed by Trichinella is important when developing immunological detection methods and vaccines and studying its interactions with the host. The present study uses the Pichia pastoris expression system to produce a soluble TbCLP antigen which induces strong antibody responses in the host during natural infection. Our results demonstrate the feasibility of TbCLP antigen production in yeasts, which are able to carry out post-translational modifications such as glycosylation and disulfide bond formation; they also indicate that the glycosylated TbCLP antigen had immunogenic effects in the tested mice and induced a mixed Th1/Th2 response, and was associated with a reduced larval burden after challenge with T. britovi. Subsequent in vitro stimulation of mice splenocytes revealed that TbCLP most likely possesses immunomodulatory properties and may play a significant role in the early phase of infection, affecting host immunological responses.

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Section: Discussionsupporting

confidence: 90%

The Immunological Properties of Recombinant Multi-Cystatin-Like Domain Protein From Trichinella Britovi Produced in Yeast

et al. 2019

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“…Moreover, human IDS have been expressed in P. pastoris , with a molecular mass larger than the one reported for the enzyme produced in mammalian cells [16]. The change in the molecular mass identified in this work could be due to hypermannosylation events, which are commonly observed in proteins expressed in P. pastoris [37], and to the fact that IDS sequence contains eight putative N-linked glycosylation sites [38].…”

Section: Discussionmentioning

confidence: 61%

Identification of the iduronate-2-sulfatase proteome in wild-type mouse brain

Cardona

Benincore

Pimentel

et al. 2019

Heliyon

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Iduronate-2-sulfatase (IDS) is a lysosomal enzyme involved in the metabolism of the glycosaminoglycans heparan (HS) and dermatan (DS) sulfate. Mutations on IDS gene produce mucopolysaccharidosis II (MPS II), characterized by the lysosomal accumulation of HS and DS, leading to severe damage of the central nervous system (CNS) and other tissues. In this study, we used a neurochemistry and proteomic approaches to identify the brain distribution of IDS and its interacting proteins on wild-type mouse brain. IDS immunoreactivity showed a robust staining throughout the entire brain, suggesting an intracellular reactivity in nerve cells and astrocytes . By using affinity purification and mass spectrometry we identified 187 putative IDS partners-proteins, mainly hydrolases, cytoskeletal proteins, transporters, transferases, oxidoreductases, nucleic acid binding proteins, membrane traffic proteins, chaperons and enzyme modulators, among others. The interactions with some of these proteins were predicted by using bioinformatics tools and confirmed by co-immunoprecipitation analysis and Blue Native PAGE. In addition, we identified cytosolic IDS-complexes containing proteins from predicted highly connected nodes (hubs), with molecular functions including catalytic activity, redox balance, binding, transport, receptor activity and structural molecule activity. The proteins identified in this study would provide new insights about IDS physiological role into the CNS and its potential role in the brain-specific protein networks.

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“…Post-translational modification for addition of glycosyl residues is a crucial step for glycoproteins including human erythropoietin (hEPO), an important molecule for promoting erythrocyte generation (Walsh and Jefferis 2006). Native hEPO which has a variety of glycosyl conjugated with maximum of 3 N-link glycosyl groups presents as isoforms with broad bands around 37 kDa (Browne et al 1986;Teh et al 2011;Jez et al 2013). As glycosylation is important to enhance its bioavailabilty, EPO molecule has been modified to bear 5 N-link glycosyl residues which known as darbepoetin (DPO) (Egrie and Browne 2001;Darling et al 2002;Fan 2015;Falck et al 2017).…”

Section: Introductionmentioning

confidence: 99%

Glycoengineering of Darbepoetin-α in CHO-DG44 Cells through Overexpression of α-2,3-sialyl-transferase and CMP-sialic Acid Transporter

et al. 2022

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Sialic acid plays a very important role in determining the circulation life span of glycoprotein in various organisms. Therefore, having a high content of sialic acid is needed by glycoprotein therapeutic agents to be able to function as desired. For example, Darbepoetin (DPO), the 5 N-linked erythropoietin showed higher bioavailability and efficacy compared to 3 N-linked erythropoietin. However, in the DPO production process, the molecular weight can vary and is highly dependent on the content of sialic acid and its production host. To improve the DPO sialic acid contents in our CHO-DG44 expressing DPO, we have engineered the cells through overexpression of α-2,3-sialyl-transferase (ST) and CMP-sialic acid transporter (CST). The DPO contained in the supernatant of the engineered cells was analyzed by Western blot and characterized by using PNGase-F or neuraminidase enzyme digestions. The results showed that, two clones, overexpressing ST or CST, were obtained. The clones showed higher molecular weight of DPO as compared to DPO expressed by the parental cells, yet retained the same protein backbone. The overexpression of these two genes does not affect cell growth. This suggests that may be these cells beneficial for therapeutic glycoproteins.

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Expression and analysis of the glycosylation properties of recombinant human erythropoietin expressed in Pichia pastoris

Cited by 17 publications

References 31 publications

The Immunological Properties of Recombinant Multi-Cystatin-Like Domain Protein From Trichinella Britovi Produced in Yeast

The Immunological Properties of Recombinant Multi-Cystatin-Like Domain Protein From Trichinella Britovi Produced in Yeast

Identification of the iduronate-2-sulfatase proteome in wild-type mouse brain

Glycoengineering of Darbepoetin-α in CHO-DG44 Cells through Overexpression of α-2,3-sialyl-transferase and CMP-sialic Acid Transporter

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