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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 ?(1,2)-xylose and core ?(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 ?(1,2)-xylosyltranferase (XylT) and the ?(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 ?(1,2)-xylose and/or ?(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.

SUBMITTER: Hanania U

PROVIDER: S-EPMC5552476 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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

Hanania Uri U Ariel Tami T Tekoah Yoram Y Fux Liat L Sheva Maor M Gubbay Yehuda Y Weiss Mara M Oz Dina D Azulay Yaniv Y Turbovski Albina A Forster Yehava Y Shaaltiel Yoseph Y

Plant biotechnology journal 20170303 9

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)-fucosyltrans ...[more]

PMID: 28160363

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Project description:BACKGROUND: Chloroplast transformation in tobacco has been used extensively to produce recombinant proteins and enzymes. Chloroplast expression cassettes can be designed with different configurations of the cis-acting elements that govern foreign gene expression. With the aim to optimize production of recombinant hemicellulases in transplastomic tobacco, we developed a set of cassettes that incorporate elements known to facilitate protein expression in chloroplasts and examined expression and accumulation of a bacterial xylanase XynA. Biomass production is another important factor in achieving sustainable and high-volume production of cellulolytic enzymes. Therefore, we compared productivity of two tobacco cultivars - a low-alkaloid and a high-biomass - as transplastomic expression platforms. RESULTS: Four different cassettes expressing XynA produced various mutant phenotypes of the transplastomic plants, affected their growth rate and resulted in different accumulation levels of the XynA enzyme. The most productive cassette was identified and used further to express XynA and two additional fungal xylanases, Xyn10A and Xyn11B, in a high-biomass tobacco cultivar. The high biomass cultivar allowed for a 60% increase in XynA production per plant. Accumulation of the fungal enzymes reached more than 10-fold higher levels than the bacterial enzyme, constituting up to 6% of the total soluble protein in the leaf tissue. Use of a well-characterized translational enhancer with the selected expression cassette revealed inconsistent effects on accumulation of the recombinant xylanases. Additionally, differences in the enzymatic activity of crude plant extracts measured in leaves of different age suggest presence of a specific xylanase inhibitor in the green leaf tissue. CONCLUSION: Our results demonstrate the pivotal importance of the expression cassette design and appropriate tobacco cultivar for high-level transplastomic production of recombinant proteins.

Dataset Information

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

Publications

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

Similar Datasets

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