Cytoprotective Effect of Recombinant Human Erythropoietin Produced in Transgenic Tobacco Plants

Kittur, Farooqahmed S.; Bah, Mamudou; Archer-Hartmann, Stephanie; Hung, Chiu-Yueh; Azadi, Parastoo; Ishihara, Mayumi; Sane, David C.; Xie, Jiahua

doi:10.1371/journal.pone.0076468

Cited by 23 publications

(49 citation statements)

References 70 publications

(97 reference statements)

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“…It has been reported that EPO glycoforms produced from tobacco showed a 2-fold increase in cytoprotective effect compared with that produced from CHO cells. Glycan analysis of the EPO expressed in tobacco cells revealed a significant amount of high-mannose EPO (32). In this perspective, the homogeneous …”

Section: Discussionmentioning

confidence: 87%

Mammalian α-1,6-Fucosyltransferase (FUT8) Is the Sole Enzyme Responsible for the N-Acetylglucosaminyltransferase I-independent Core Fucosylation of High-mannose N-Glycans

Yang

Wang

2016

Journal of Biological Chemistry

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Understanding the biosynthetic pathway of protein glycosylation in various expression cell lines is important for controlling and modulating the glycosylation profiles of recombinant glycoproteins. We found that expression of erythropoietin (EPO) in a HEK293S N-acetylglucosaminyltransferase I (GnT I) ؊/؊ cell line resulted in production of the Man 5 GlcNAc 2 glycoforms, in which more than 50% were core-fucosylated, implicating a clear GnT I-independent core fucosylation pathway. Expression of GM-CSF and the ectodomain of Fc␥IIIA receptor led to ϳ30% and 3% core fucosylation, suggesting that the level of core fucosylation also depends on the nature of the recombinant proteins.To elucidate the GnT I-independent core fucosylation pathway, we generated a stable HEK293S GnT I ؊/؊ cell line with either knockdown or overexpression of FUT8 by a highly efficient lentivirus-mediated gene transfer approach. We found that the EPO produced from the FUT8 knockdown cell line was the pure Man 5 GlcNAc 2 glycoform, whereas that produced from the FUT8-overexpressing cell line was found to be fully core-fucosylated oligomannose glycan (Man 5 GlcNAc 2 Fuc). These results provide direct evidence that FUT8, the mammalian ␣1,6-fucosyltransferase, is the sole enzyme responsible for the GnT I-independent core fucosylation pathway. The production of the homogeneous core-fucosylated Man 5 GlcNAc 2 glycoform of EPO in the FUT8-overexpressed HEK293S GnT I ؊/؊ cell line represents the first example of production of fully core-fucosylated high-mannose glycoforms.

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Section: Discussionmentioning

confidence: 87%

Mammalian α-1,6-Fucosyltransferase (FUT8) Is the Sole Enzyme Responsible for the N-Acetylglucosaminyltransferase I-independent Core Fucosylation of High-mannose N-Glycans

Yang

Wang

2016

Journal of Biological Chemistry

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“…Leaf tissues harvested from greenhouse grown transgenic tobacco line A56-5 with high expression of asialo-rhuEPO P (Kittur et al 2013) were used for DNA, RNA and protein purification throughout the studies unless otherwise stated.…”

Section: Methodsmentioning

confidence: 99%

“…The asialo-rhuEPO P peak fractions were collected and further purified using an IAC column. Asialo-rhuEPO P in each IAC fraction was determined using a sandwich ELISA as described previously (Kittur et al 2013). Residual BSA in the purified fraction was removed using Melon ™ gel BSA removal kit (Thermo Scientific, Waltham, MA, USA).…”

Section: Methodsmentioning

confidence: 99%

“…Recent studies have also shown that asialo-rhuEPO can benefit diabetes patients by promoting β-cell growth (Choi et al 2010). Since the current method for the production of asialo-rhuEPO from rhuEPO M by enzymatic desialylation is not economically viable, we (Kittur et al 2012; 2013) and Parson et al (2012) stably co-expressed human EPO and β1,4-galactosyltransferase ( GalT ) genes in tobacco plants and moss, respectively to produce asialo-rhuEPO P . These studies have demonstrated that asialo-rhuEPO can accumulate at high levels in transgenic plants (Parson et al 2012; Kittur et al 2013), and that plants could be used as a bioreactor for the production of this medically important glycoprotein.…”

Section: Introductionmentioning

confidence: 99%

“…In our recent asialo-rhuEPO expression study (Kittur et al 2013), EPO protein was appended with a C -terminal TEV protease cleavage site, a Strep -tag II, followed by an endoplasmic reticulum (ER) retention signal KDEL. Western blot analysis of transgenic tobacco plant leaf extracts showed high levels of asialo-rhuEPO P .…”

Section: Introductionmentioning

confidence: 99%

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C-Terminally fused affinity Strep-tag II is removed by proteolysis from recombinant human erythropoietin expressed in transgenic tobacco plants

et al. 2014

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Asialo-erythropoietin (asialo-EPO), a desialylated form of EPO, is a potent tissue-protective agent. Recently, we and others have exploited a low cost plant-based expression system to produce recombinant human asialo-EPO (asialo-rhuEPOP). To facilitate purification from plant extracts, Strep-tag II was engineered at the C-terminus of EPO. Although asialo-rhuEPOP was efficiently expressed in transgenic tobacco plants, affinity purification based on Strep-tag II did not result in the recovery of the protein. In this study, we investigated the stability of Strep-tag II tagged asialo-rhuEPOP expressed in tobacco plants to understand whether this fused tag is cleaved or inaccessible. Sequencing RT-PCR products confirmed that fused DNA sequences encoding Strep-tag II were properly transcribed, and three-dimensional protein structure model revealed that the tag must be fully accessible. However, Western blot analysis of leaf extracts and purified asialo-rhuEPOP revealed that the Strep-tag II was absent on the protein. Additionally, no peptide fragment containing Strep-tag II was identified in the LC-MS/MS analysis of purified protein further supporting that the affinity tag was absent on asialo-rhuEPOP. However, Strep-tag II was detected on asialo-rhuEPOP that was retained in the endoplasmic reticulum, suggesting that the Strep-tag II is removed during protein secretion or extraction. These findings together with recent reports that C-terminally fused Strep-tag II or IgG Fc domain are also removed from EPO in tobacco plants, suggest that its C-terminus may be highly susceptible to proteolysis in tobacco plants. Therefore, direct fusion of purification tags at the C-terminus of EPO should be avoided while expressing it in tobacco plants.

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Basic leucine zipper transcription activators – tools to improve production and quality of human erythropoietin in Nicotiana benthamiana

Wagner,

Musiychuk,

Shoji

et al. 2024

Biotechnology Journal

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Human erythropoietin (hEPO) is one of the most in‐demand biopharmaceuticals, however, its production is challenging. When produced in a plant expression system, hEPO results in extensive plant tissue damage and low expression. It is demonstrated that the modulation of the plant protein synthesis machinery enhances hEPO production. Co‐expression of basic leucine zipper transcription factors with hEPO prevents plant tissue damage, boosts expression, and increases hEPO solubility. bZIP28 co‐expression up‐regulates genes associated with the unfolded protein response, indicating that the plant tissue damage caused by hEPO expression is due to the native protein folding machinery being overwhelmed and that this can be overcome by co‐expressing bZIP28.

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Cytoprotective Effect of Recombinant Human Erythropoietin Produced in Transgenic Tobacco Plants

Cited by 23 publications

References 70 publications

Mammalian α-1,6-Fucosyltransferase (FUT8) Is the Sole Enzyme Responsible for the N-Acetylglucosaminyltransferase I-independent Core Fucosylation of High-mannose N-Glycans

Mammalian α-1,6-Fucosyltransferase (FUT8) Is the Sole Enzyme Responsible for the N-Acetylglucosaminyltransferase I-independent Core Fucosylation of High-mannose N-Glycans

C-Terminally fused affinity Strep-tag II is removed by proteolysis from recombinant human erythropoietin expressed in transgenic tobacco plants

Basic leucine zipper transcription activators – tools to improve production and quality of human erythropoietin in Nicotiana benthamiana

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