Establishment of a tobacco BY2 cell line devoid of plant-specific xylose and fucose as a platform for the production of biotherapeutic proteins
| Autor: | Yoseph Shaaltiel, Yehuda Gubbay, Yoram Tekoah, Mara Weiss, Albina Turbovski, Dina Oz, Liat Fux, Maor Sheva, Yehava Forster, Tami Ariel, Yaniv Azulay, Uri Hanania |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Glycan Nicotiana tabacum Genetic Vectors Plant Science Biology Fucose Cell Line law.invention DNaseI Fucosyltransferases 03 medical and health sciences chemistry.chemical_compound Polysaccharides law Tobacco Clustered Regularly Interspaced Short Palindromic Repeats Pentosyltransferases CRISPR/Cas9 Gene Research Articles plant glycans Glycoproteins Plant Proteins glyco‐engineering Gene Editing chemistry.chemical_classification Xylose bio‐pharming biology.organism_classification Recombinant Proteins Biological Therapy 030104 developmental biology Biochemistry chemistry Cell culture Recombinant DNA biology.protein Glycoprotein Agronomy and Crop Science Research Article Biotechnology |
| Zdroj: | Plant Biotechnology Journal |
| ISSN: | 1467-7644 |
| Popis: | Summary Plant‐produced glycoproteins contain N‐linked glycans with plant‐specific residues of β(1,2)‐xylose and core α(1,3)‐fucose, which do not exist in mammalian‐derived proteins. Although our experience with two enzymes that are used for enzyme replacement therapy does not indicate that the plant sugar residues have deleterious effects, we made a conscious decision to eliminate these moieties from plant‐expressed proteins. We knocked out the β(1,2)‐xylosyltranferase (XylT) and the α(1,3)‐fucosyltransferase (FucT) genes, using CRISPR/Cas9 genome editing, in Nicotiana tabacum L. cv Bright Yellow 2 (BY2) cell suspension. In total, we knocked out 14 loci. The knocked‐out lines were stable, viable and exhibited a typical BY2 growing rate. Glycan analysis of the endogenous proteins of these lines exhibited N‐linked glycans lacking β(1,2)‐xylose and/or α(1,3)‐fucose. The knocked‐out lines were further transformed successfully with recombinant DNaseI. The expression level and the activity of the recombinant protein were similar to that of the protein produced in the wild‐type BY2 cells. The recombinant DNaseI was shown to be totally free from any xylose and/or fucose residues. The glyco‐engineered BY2 lines provide a valuable platform for producing potent biopharmaceutical products. Furthermore, these results demonstrate the power of the CRISPR/Cas9 technology for multiplex gene editing in BY2 cells. |
| Databáze: | OpenAIRE |
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