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Influence of the KDEL signal, DMSO and mannitol on the production of the recombinant antibody 14D9 by long-term Nicotiana tabacum cell suspension culture

Abstract

We have established two transgenic cell suspension culture lines of Nicotiana tabacum that express the catalytic antibody 14D9 as a secretory product (sec-Ab) or as a KDEL-tagged product in the endoplasmic reticulum (Ab-KDEL), respectively. After 3 years of culture, the performance improved to a production level of 0.15 ± 0.03 μg ml−1 on the seventh day of culture for the sec-Ab line and 0.48 ± 0.05 μg ml−1 on the third day for Ab-KDEL line. Analysis of the effect of osmotic stress using mannitol (90 g l−1) as an osmolite revealed that there was a 12-fold increase in antibody yield (1.96 ± 0.20 μg ml−1) on the seventh day of culture in line sec-Ab and a fivefold increase (2.31 ± 0.18 μg ml−1) on the seventh day for line Ab-KDEL. The concentration of the antibody in the culture medium was not significant. Dimethyl sulfoxide used as a permeabilizing agent was not effective in increasing 14D9 yield, but it did cause distinctive cell damage at all concentrations tested.

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Abbreviations

DMSO:

Dimethyl sulfoxide

DW:

Dry weight

ER:

Endoplasmic reticulum

FW:

Fresh weight

GI:

Growth index

μ:

Specific growth rate

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Acknowledgements

This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) from Argentina (PIP5578) and the Agencia Nacional de Producción Científica y Tecnológica (ANPCyT) (PICT2007 0552). Drs. P. Marconi, S.Petruccelli and M.A. Alvarez belong to CONICET. Julieta López is a PhD student (ANPCyT). We wish to thank Ms. Carlota Thompson for her advice and careful revision of English.

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Correspondence to M. A. Alvarez.

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López, J., Lencina, F., Petruccelli, S. et al. Influence of the KDEL signal, DMSO and mannitol on the production of the recombinant antibody 14D9 by long-term Nicotiana tabacum cell suspension culture. Plant Cell Tiss Organ Cult 103, 307–314 (2010). https://doi.org/10.1007/s11240-010-9780-y

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Keywords

  • Molecular farming
  • 14D9
  • In vitro plant cell cultures
  • Recombinant proteins