Dataset Information

Cytoprotective effect of recombinant human erythropoietin produced in transgenic tobacco plants.

ABSTRACT: Asialo-erythropoietin, a desialylated form of human erythropoietin (EPO) lacking hematopoietic activity, is receiving increased attention because of its broader protective effects in preclinical models of tissue injury. However, attempts to translate its protective effects into clinical practice is hampered by unavailability of suitable expression system and its costly and limit production from expensive mammalian cell-made EPO (rhuEPO(M)) by enzymatic desialylation. In the current study, we took advantage of a plant-based expression system lacking sialylating capacity but possessing an ability to synthesize complex N-glycans to produce cytoprotective recombinant human asialo-rhuEPO. Transgenic tobacco plants expressing asialo-rhuEPO were generated by stably co-expressing human EPO and ?1,4-galactosyltransferase (GalT) genes under the control of double CaMV 35S and glyceraldehyde-3-phosphate gene (GapC) promoters, respectively. Plant-produced asialo-rhuEPO (asialo-rhuEPO(P)) was purified by immunoaffinity chromatography. Detailed N-glycan analysis using NSI-FTMS and MS/MS revealed that asialo-rhuEPO(P) bears paucimannosidic, high mannose-type and complex N-glycans. In vitro cytoprotection assays showed that the asialo-rhuEPO(P) (20 U/ml) provides 2-fold better cytoprotection (44%) to neuronal-like mouse neuroblastoma cells from staurosporine-induced cell death than rhuEPO(M) (21%). The cytoprotective effect of the asialo-rhuEPO(P) was found to be mediated by receptor-initiated phosphorylation of Janus kinase 2 (JAK2) and suppression of caspase 3 activation. Altogether, these findings demonstrate that plants are a suitable host for producing cytoprotective rhuEPO derivative. In addition, the general advantages of plant-based expression system can be exploited to address the cost and scalability issues related to its production.

SUBMITTER: Kittur FS

PROVIDER: S-EPMC3790672 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Cytoprotective effect of recombinant human erythropoietin produced in transgenic tobacco plants.

Kittur Farooqahmed S FS Bah Mamudou M Archer-Hartmann Stephanie S Hung Chiu-Yueh CY Azadi Parastoo P Ishihara Mayumi M Sane David C DC Xie Jiahua J

PloS one 20131004 10

Asialo-erythropoietin, a desialylated form of human erythropoietin (EPO) lacking hematopoietic activity, is receiving increased attention because of its broader protective effects in preclinical models of tissue injury. However, attempts to translate its protective effects into clinical practice is hampered by unavailability of suitable expression system and its costly and limit production from expensive mammalian cell-made EPO (rhuEPO(M)) by enzymatic desialylation. In the current study, we too ...[more]

PMID: 24124563

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Project description:KEY MESSAGE:C -terminally fused Strep -tag II is removed from rhuEPO expressed in tobacco plants. The finding suggests that direct fusion of purification tags at the C -terminus of rhuEPO should be avoided. 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-rhuEPO(P)). To facilitate purification from plant extracts, Strep-tag II was engineered at the C-terminus of EPO. Although asialo-rhuEPO(P) 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-rhuEPO(P) 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-rhuEPO(P) 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-rhuEPO(P). However, Strep-tag II was detected on asialo-rhuEPO(P) 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.

Project description:Transgenic plants have the potential to produce recombinant proteins on an agricultural scale, with yields of several tons per year. The cost-effectiveness of transgenic plants increases if simple cultivation facilities such as greenhouses can be used for production. In such a setting, we expressed a novel affinity ligand based on the fluorescent protein DsRed, which we used as a carrier for the linear epitope ELDKWA from the HIV-neutralizing antibody 2F5. The DsRed-2F5-epitope (DFE) fusion protein was produced in 12 consecutive batches of transgenic tobacco (Nicotiana tabacum) plants over the course of 2 years and was purified using a combination of blanching and immobilized metal-ion affinity chromatography (IMAC). The average purity after IMAC was 57 ± 26% (n = 24) in terms of total soluble protein, but the average yield of pure DFE (12 mg kg-1) showed substantial variation (± 97 mg kg-1, n = 24) which correlated with seasonal changes. Specifically, we found that temperature peaks (>28°C) and intense illuminance (>45 klx h-1) were associated with lower DFE yields after purification, reflecting the loss of the epitope-containing C-terminus in up to 90% of the product. Whereas the weather factors were of limited use to predict product yields of individual harvests conducted for each batch (spaced by 1 week), the average batch yields were well approximated by simple linear regression models using two independent variables for prediction (illuminance and plant age). Interestingly, accumulation levels determined by fluorescence analysis were not affected by weather conditions but positively correlated with plant age, suggesting that the product was still expressed at high levels, but the extreme conditions affected its stability, albeit still preserving the fluorophore function. The efficient production of intact recombinant proteins in plants may therefore require adequate climate control and shading in greenhouses or even cultivation in fully controlled indoor farms.

Dataset Information

Cytoprotective effect of recombinant human erythropoietin produced in transgenic tobacco plants.

Publications

Cytoprotective effect of recombinant human erythropoietin produced in transgenic tobacco plants.

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