Production of Recombinant Glycoproteins in Nicotiana tabacum BY-2 Suspension Cells

Navarre, Catherine; Chaumont, François

doi:10.1007/978-1-0716-2241-4_5

Cited by 5 publications

(3 citation statements)

References 30 publications

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“…The N. tabacum cv. Bright Yellow 2 (BY‐2) suspension cells (Nagata et al, 1992) were transformed by Agrobacterium tumefaciens and the transformed lines selected on MS medium [4.4 g/L Murashige and Skoog salts (MP Biomedicals, 2610024), 30 g/L sucrose, 0.2 g/L KH 2 PO 4 , 2.5 mg/L thiamine, 50 mg/L myo‐inositol and 0.2 mg/L 2,4‐dichlorophenoxyacetic acid, pH 5.8 (KOH)] containing cefotaxime (500 mg/mL), carbenicillin (500 mg/mL) and kanamycin (100 mg/mL) as described (Navarre & Chaumont, 2022). Liquid culture of BY‐2 suspension cells (50 mL of medium in a 250 mL Erlenmeyer flask) were grown in the dark at 25°C with agitation on a rotary shaker (90 rpm).…”

Section: Methodsmentioning

confidence: 99%

A new method to measure aquaporin‐facilitated membrane diffusion of hydrogen peroxide and cations in plant suspension cells

Ahmed,

Ismail,

Ding

et al. 2023

Plant Cell & Environment

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Plant aquaporins (AQPs) facilitate the membrane diffusion of water and small solutes, including hydrogen peroxide (H2O2) and, possibly, cations, essential signalling molecules in many physiological processes. While the determination of the channel activity generally depends on heterologous expression of AQPs in Xenopus oocytes or yeast cells, we established a genetic tool to determine whether they facilitate the diffusion of H2O2 through the plasma membrane in living plant cells. We designed genetic constructs to co‐express the fluorescent H2O2 sensor HyPer and AQPs, with expression controlled by a heat shock‐inducible promoter in Nicotiana tabacum BY‐2 suspension cells. After induction of ZmPIP2;5 AQP expression, a HyPer signal was recorded when the cells were incubated with H2O2, suggesting that ZmPIP2;5 facilitates H2O2 transmembrane diffusion; in contrast, the ZmPIP2;5W85A mutated protein was inactive as a water or H2O2 channel. ZmPIP2;1, ZmPIP2;4 and AtPIP2;1 also facilitated H2O2 diffusion. Incubation with abscisic acid and the elicitor flg22 peptide induced the intracellular H2O2 accumulation in BY‐2 cells expressing ZmPIP2;5. We also monitored cation channel activity of ZmPIP2;5 using a novel fluorescent photo‐switchable Li+ sensor in BY‐2 cells. BY‐2 suspension cells engineered for inducible expression of AQPs as well as HyPer expression and the use of Li+ sensors constitute a powerful toolkit for evaluating the transport activity and the molecular determinants of PIPs in living plant cells.

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Section: Methodsmentioning

confidence: 99%

A new method to measure aquaporin‐facilitated membrane diffusion of hydrogen peroxide and cations in plant suspension cells

Ahmed,

Ismail,

Ding

et al. 2023

Plant Cell & Environment

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“…Agrobacterium tumefaciens LBA4404VirG‐mediated stable transformation of BY‐2 cells was carried out as described by Navarre and Chaumont ( 2022 ). nptII resistance marker was used to transform WT and XylT / FucT ‐KO BY‐2 cell lines, hpt resistance gene was used to transform GnTI ‐KO and GnTI / FucT ‐KO ( GnTI (/ FucT )‐KO) cell lines.…”

Section: Methodsmentioning

confidence: 99%

In vivo deglycosylation of recombinant glycoproteins in tobacco BY‐2 cells

Herman

Far

Peeters

et al. 2023

Plant Biotechnology Journal

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SummaryProduction of recombinant pharmaceutical glycoproteins has been carried out in multiple expression systems. However, N‐glycosylation, which increases heterogeneity and raises safety concerns due to the presence of non‐human residues, is usually not controlled. The presence and composition of N‐glycans are also susceptible to affect protein stability, function and immunogenicity. To tackle these issues, we are developing glycoengineered Nicotiana tabacum Bright Yellow‐2 (BY‐2) cell lines through knock out and ectopic expression of genes involved in the N‐glycosylation pathway. Here, we report on the generation of BY‐2 cell lines producing deglycosylated proteins. To this end, endoglycosidase T was co‐expressed with an immunoglobulin G or glycoprotein B of human cytomegalovirus in BY‐2 cell lines producing only high mannose N‐glycans. Endoglycosidase T cleaves high mannose N‐glycans to generate single, asparagine‐linked, N‐acetylglucosamine residues. The N‐glycosylation profile of the secreted antibody was determined by mass spectrometry analysis. More than 90% of the N‐glycans at the conserved Asn297 site were deglycosylated. Likewise, extensive deglycosylation of glycoprotein B, which possesses 18 N‐glycosylation sites, was observed. N‐glycan composition of gB glycovariants was assessed by in vitro enzymatic mobility shift assay and proven to be consistent with the expected glycoforms. Comparison of IgG glycovariants by differential scanning fluorimetry revealed a significant impact of the N‐glycosylation pattern on the thermal stability. Production of deglycosylated pharmaceutical proteins in BY‐2 cells expands the set of glycoengineered BY‐2 cell lines.

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“…The XylT/FucT KO BY-2 cell line, knocked out for genes encoding b(1,2)-xylosyltransferases and a(1,3)fucosyltransferases (Mercx et al, 2017), was used to generate transgenic cell lines expressing gP via A. tumefaciens LBA4404VirG-mediated stable transformation of BY-2 cells as described (Navarre and Chaumont, 2022). Biolistic particle delivery in BY-2 cells was carried out with a Biolistic PDS1000/He device (Bio-Rad, Hercules, CA, USA) as previously described (Herman et al, 2021).…”

Section: Transformation Of By-2 Cellsmentioning

confidence: 99%

Issue when expressing a recombinant protein under the control of p35S in Nicotiana tabacum BY-2 cells

Navarre,

Orval,

Peeters

et al. 2023

Front. Plant Sci.

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Several recombinant proteins have been successfully produced in plants. This usually requires Agrobacterium-mediated cell transformation to deliver the T-DNA into the nucleus of plant cells. However, some genetic instability may threaten the integrity of the expression cassette during its delivery via A. tumefaciens, especially when the protein of interest is toxic to the bacteria. In particular, we found that a Tn3 transposon can be transferred from the pAL4404 Ti plasmid of A. tumefaciens LBA4404 into the expression cassette when using the widely adopted 35S promoter, thereby damaging T-DNA and preventing correct expression of the gene of interest in Nicotiana tabacum BY-2 suspension cells.

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Production of Recombinant Glycoproteins in Nicotiana tabacum BY-2 Suspension Cells

Cited by 5 publications

References 30 publications

A new method to measure aquaporin‐facilitated membrane diffusion of hydrogen peroxide and cations in plant suspension cells

A new method to measure aquaporin‐facilitated membrane diffusion of hydrogen peroxide and cations in plant suspension cells

In vivo deglycosylation of recombinant glycoproteins in tobacco BY‐2 cells

Issue when expressing a recombinant protein under the control of p35S in Nicotiana tabacum BY-2 cells

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