Establishment of a tobacco BY2 cell line devoid of plant-specific xylose and fucose as a platform for the production of biotherapeutic proteins.
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.
Protalix Biotherapeutics, Carmiel, Israel.
Uri
U
Hanania
Protalix Biotherapeutics, Carmiel, Israel.
Tami
T
Ariel
Protalix Biotherapeutics, Carmiel, Israel.
Yoram
Y
Tekoah
Protalix Biotherapeutics, Carmiel, Israel.
Liat
L
Fux
Protalix Biotherapeutics, Carmiel, Israel.
Maor
M
Sheva
Protalix Biotherapeutics, Carmiel, Israel.
Yehuda
Y
Gubbay
Protalix Biotherapeutics, Carmiel, Israel.
Mara
M
Weiss
Protalix Biotherapeutics, Carmiel, Israel.
Dina
D
Oz
Protalix Biotherapeutics, Carmiel, Israel.
Yaniv
Y
Azulay
Protalix Biotherapeutics, Carmiel, Israel.
Albina
A
Turbovski
Protalix Biotherapeutics, Carmiel, Israel.
Yehava
Y
Forster
Protalix Biotherapeutics, Carmiel, Israel.
Yoseph
Y
Shaaltiel
D001691:Biological Therapy; D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D005643:Fucose / Q000378:metabolism*; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism; D000072669:Gene Editing; D005822:Genetic Vectors; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D010430:Pentosyltransferases / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011134:Polysaccharides; D011994:Recombinant Proteins; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism; D014994:Xylose / Q000378:metabolism*; D000097608:UDP Xylose-Protein Xylosyltransferase
D005643:Fucose; D005647:Fucosyltransferases; D006023:Glycoproteins; D010430:Pentosyltransferases; D010940:Plant Proteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D014994:Xylose; D000097608:UDP Xylose-Protein Xylosyltransferase
CRISPR/Cas9; DNaseI; bio-pharming; gene editing; glyco-engineering; plant glycans