Glycoengineering tobacco plants to stably express recombinant human erythropoietin with different N-glycan profiles
| Autor: | Jiahua Xie, Clemens Gruber, Asif Shajahan, Chiu-Yueh Hung, Somanath Kallolimath, Jackson L. Pearce, Michelle D. Thomas, Farooqahmed S. Kittur, Parastoo Azadi, Chuanshu Zhu |
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| Rok vydání: | 2020 |
| Předmět: |
Glycan
Glycosylation Transgene Gene Expression 02 engineering and technology Biochemistry Article law.invention 03 medical and health sciences chemistry.chemical_compound N-linked glycosylation Polysaccharides Structural Biology law Tobacco Humans Erythropoietin Molecular Biology Gene 030304 developmental biology chemistry.chemical_classification 0303 health sciences biology General Medicine 021001 nanoscience & nanotechnology Recombinant Proteins Sialic acid Cell biology carbohydrates (lipids) chemistry Recombinant DNA biology.protein Genetic Engineering 0210 nano-technology Glycoprotein Genome Plant |
| Zdroj: | Int J Biol Macromol |
| ISSN: | 0141-8130 |
| Popis: | Plant-based expression system can be used to produce biopharmaceuticals with many potential advantages, but plants cannot be directly used to express functional human glycoproteins because of their differences in glycosylation abilities from mammals. To exploit a plant-based expression for producing recombinant human erythropoietin (rhuEPO), we glycoengineered tobacco plants by stably introducing seven to eight mammalian genes including a target human EPO into tobacco genome in order to generate capacities for β1,4-galactosylation, bisecting N-acetylglucosamine (GlcNAc) and sialylation. Wild type human β1,4-galactosyltransferase gene (GalT) or a chimeric GalT gene (ST/GalT) with GalT cytoplasmic-transmembrane-stem region (CTS) replaced by the CTS from the rat 2,6-sialyltransferase (ST) was co-expressed to produce rhuEPO bearing β1,4-galactose-extended N-glycan chains as well as to compare their β1,4-galactosylation efficiencies. Mammalian UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene (GNE), N-acetylneuraminic acid phosphate synthase gene (NANS), CMP-N-acetylneuraminic acid synthetase gene (CMAS), CMP-sialic acid transporter gene (CST) and α−2,6-sialyltransferase gene (ST) were co-expressed to build sialylation capacity in plants. In addition, the human MGAT3 encoding N-acetylglucosaminyltransferase III (GnTIII) was also co-expressed to produce N-glycan chains with bisecting GlcNAc. Our PCR and RT-PCR results demonstrated that the above transgenes were not only incorporated into tobacco genome but also properly transcribed. Both GalT and ST/GalT were found to add β1,4-galactose residues to the N-glycan chains, but the latter was more efficient. Furthermore, co-expressing MGAT3 could generate bisected GlcNAc. However, our current efforts did result in generating sialylation capacity. Created transgenic plants expressing EPO and ST/GalT could be used to produce rhuEPO with high proportion of β1,4-galactose-extended N-glycan chains for tissue protective purposes. |
| Databáze: | OpenAIRE |
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