Establishment of a tobacco BY2 cell line devoid of plant-specific xylose and fucose as a platform for the production of biotherapeutic proteins

Abstract

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.

Keywords: CRISPR/Cas9; DNaseI; bio-pharming; gene editing; glyco-engineering; plant glycans.

Figure 1

Schematic illustration of the first three exons and introns of the five FucT genes of N. Tabaccum. Exons 1, 2, 3 are shown as boxes and introns 1, 2, 3 as lines. The size of each exon and intron is indicated in base pairs (bp).

Figure 2

Schematic description of the three binary vectors used for the transformation of the BY2 cells. (a) The phCas9‐XylT; (b) the phCas9‐FucT; (c) the phCas9‐XylT/FucT. LB, left border; IRES, internal ribosome entry site; hpt, hygromycin phosphotransferase; N‐ter, nopaline synthase terminator; 35S, 35S cauliflower mosaic virus promoter with omega enhancer; hCas9, human‐optimized Cas9; NLS, SV40 nuclear localization signal; O‐ter, octopine synthase terminator; U6, Arabidopsis U6 promoter; sgRNA, chimeras of the various crRNAs with tracrRNA, the colour boxes represent the five different crRNAs that were used; RB, right border.

Figure 3

Western blots using anti‐xylose, anti‐fucose or anti‐HRP antibodies. The total soluble protein was extracted from the glyco‐engineered and the nontransgenic BY2 cells and 10 μg of protein from each sample were loaded on SDS‐PAGE followed by Western blot using (a) anti‐xylose, (b) anti‐fucose and (c) anti‐HRP antibodies. 1, ΔXT cell line; 2, ΔFT cell line; 3, ΔXFT cell line; 4, the wild‐type nontransgenic BY2 cells. MW, molecular weight marker in kDa.

Figure 4

UPLC chromatograms presenting the peaks of the main glycans derived from the glycoproteins derived from (top to bottom): BY2 control cells (WT); a XylT knocked‐out line (ΔXT); a FucT knocked‐out line (ΔFT); and a XylT/FucT knocked‐out cell line (ΔXFT). Glycans are annotated with cartoons and acronyms above main peaks. Symbols are as follows: ‐Mannose; ‐Xylose; ‐N‐acetylglucosamine; ‐Fucose. Glycan acronyms are based on the nomenclature indicated at the following website http://www.proglycan.com/sites/default/public/pdf/nomen_2007.pdf.

Figure 5

UPLC chromatograms presenting the peaks of the main glycans released from a DNaseI protein (top to bottom): Glycan pool derived from DNaseI produced by control cells (WT); glycan pool from a XylT knocked‐out line (ΔXT); glycan pool released from DNaseI produced from a FucT knocked‐out line (ΔFT); and a glycan pool released from DNaseI from a XylT/FucT knocked‐out cell line (ΔXFT). Glycans are annotated with cartoons and acronyms above main peaks. Symbols are as follows: ‐Mannose; ‐Xylose; ‐N‐acetylglucosamine; ‐Fucose. Glycan acronyms are based on the nomenclature indicated at the following website http://www.proglycan.com/sites/default/public/pdf/nomen_2007.pdf.