Repositorium
Establishment of a tobacco BY2 cell line devoid of plant-specific xylose and fucose as a platform for the production of biotherapeutic proteins
67
Journal Article / 2017
Hanania, Uri; Ariel, Tami; Tekoah, Yoram; Fux, Liat; Sheva, Maor; Gubbay, Yehuda; Weiss, Mara; Oz, Dina; Azulay, Yaniv; Turbovski, Albina; Forster, Yehava; Shaaltiel, Yoseph
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.
Techniques
ID | Corresponding Author Country |
Plant Species | GE Technique Sequence Identifier |
Trait Type of Alteration |
Progress in Research Key Topic |
---|---|---|---|---|---|
135 |
Tekoah, Yoram Israel |
Nicotiana benthamiana |
CRISPR/Cas9 FuCT |
improved capacity to produce glycoproteins SDN1 |
Basic research Basic research |
136 |
Tekoah, Yoram Israel |
Nicotiana benthamiana |
CRISPR/Cas9 XylT |
improved capacity to produce glycoproteins SDN1 |
Basic research Basic research |
137 |
Tekoah, Yoram Israel |
Nicotiana benthamiana |
CRISPR/Cas9 XylT/ FuCT |
improved capacity to produce glycoproteins SDN1 |
Basic research Basic research |