The expression of the 14D9 catalytic antibody in suspended cells of<i>Nicotiana tabacum</i>cultures increased by the addition of protein stabilizers and by transference from erlenmeyer flasks to a 2‐L bioreactor

Marconi, Patricia Laura; Álvarez, María Alejandra

doi:10.1002/btpr.1940

Cited by 7 publications

(4 citation statements)

References 27 publications

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“…It is noteworthy there were only a few cleaved EGFP products (day 0 to 12) and no 45 kDa intermediate was detected in the media by the anti‐EGFP Western blotting. These results indicate: (i) the secreted fusion glycoprotein is relatively stable in media, which is attributed to the low levels of protease activity presence in media as well as the steric protection imparted by the Hyp‐glycans; (ii) Hyp‐ O ‐glycosylation of the (SP) 32 tag is a two‐step process with the first step involving constitutive addition of two or three galactose to the Hyp residue (partial glycosylation), followed by a second critical rate‐limiting step triggering the cascade for addition of the other Hyp‐glycan residues (full glycosylation); (iii) The full glycosylation must be essential in directing effective secretion of the recombinant protein, and enzymatically building the complex arabinogalactan structure onto the initially di‐ or tri‐galactosylated (SP) 32 ‐EGFP intermediate, once triggered, is highly efficient inside cells. This was also reflected in the observation of the relatively higher accumulation level of this intermediate on day 2 (before the full glycosylation was triggered) in relation to the rest of days of culture (Fig.…”

Section: Resultsmentioning

confidence: 99%

High‐yield secretion of recombinant proteins expressed in tobacco cell culture with a designer glycopeptide tag: Process development

González

Savary

2015

Biotechnology Journal

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Low-yield protein production remains the most significant economic hurdle with plant cell culture technology. Fusions of recombinant proteins with hydroxyproline-O-glycosylated designer glycopeptide tags have consistently boosted secreted protein yields. This prompted us to study the process development of this technology aiming to achieve productivity levels necessary for commercial viability. We used a tobacco BY-2 cell culture expressing EGFP as fusion with a glycopeptide tag comprised of 32 repeat of "Ser-Pro" dipeptide, or (SP)32 , to study cell growth and protein secretion, culture scale-up, and establishment of perfusion cultures for continuous production. The BY-2 cells accumulated low levels of cell biomass (~7.5 g DW/L) in Schenk & Hildebrandt medium, but secreted high yields of (SP)32 -tagged EGFP (125 mg/L). Protein productivity of the cell culture has been stable for 6.0 years. The BY-2 cells cultured in a 5-L bioreactor similarly produced high secreted protein yield at 131 mg/L. Successful operation of a cell perfusion culture for 30 days was achieved under the perfusion rate of 0.25 and 0.5 day(-1) , generating a protein volumetric productivity of 17.6 and 28.9 mg/day/L, respectively. This research demonstrates the great potential of the designer glycopeptide technology for use in commercial production of valuable proteins with plant cell cultures.

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Section: Resultsmentioning

confidence: 99%

High‐yield secretion of recombinant proteins expressed in tobacco cell culture with a designer glycopeptide tag: Process development

González

Savary

2015

Biotechnology Journal

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“…It has excellent properties for PMF, such as metabolic inertness, low cost, and easy separation along the DPS, unlike other polymers, such as bovine serum albumin [90]. In addition, PVPs do not affect root growth or RP yield in biomass [25,27,40,58]. In its best performances, PVP showed a 43% increase in the ratio of RP rhizosecretion.TSP − 1 and held protein degradation at minimum rates during 46 days of culture [7,44].…”

Section: Rhizosecretion Performancementioning

confidence: 99%

“…The root system has the bene ts of speed, high scalability, and low risk of contamination with human pathogens. Furthermore, several reports show the root-based recombinant expression as more productive than the leaf system [22][23][24][25][26].…”

Section: Introductionmentioning

confidence: 99%

Nicotiana hairy roots for recombinant protein expression, where to start? A systematic review

et al. 2023

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Hairy roots are a plant-tissue culture raised by Rhizobium rhizogenes infection (formerly known as Agrobacterium rhizogenes). Nowadays, these roots have been gaining more space in biotechnology due to their bene ts for the recombinant expression of valuables proteins; it includes simpli ed downstream processing, protein rhizosecretion, and scalability in bioreactors. However, due to methodological inconsistency among reports, the tissue platform is still a disruptive technology. In the current paper, we propose the rst step to overcome this issue through a systematic review of studies that employ Nicotiana hairy roots for recombinant expression. We conducted a qualitative synthesis of 36 out of 387 publications initially selected. Following the PRISMA procedure, all papers were assessed for exclusion and inclusion criteria. Multiple points of root culture were explored, including transformation methods, root growth curve, external additives, and scale-up with bioreactors to determine which approaches performed best and what is still required to achieve a robust protocol. The information presented here may help researchers who want to work with hairy roots in their laboratories trace a successful path to high recombinant expression.

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Section: Introductionmentioning

confidence: 99%

Nicotiana Hairy Roots for Recombinant Protein Expression, Where to Start? A systematic review

Aragão

Álvarez

Caiafa

et al. 2022

Preprint

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Hairy roots are a plant-tissue culture raised by Rhizobium rhizogenes infection (formerly known as Agrobacterium rhizogenes). Nowadays, these roots have been gaining more space in biotechnology due to their benefits for the recombinant expression of valuables proteins; it includes simplified downstream processing, protein rhizosecretion, and scalability in bioreactors. However, due to methodological inconsistency among reports, the tissue platform is still a disruptive technology. In the current paper, we propose the first step to overcome this issue through a systematic review of studies that employ Nicotiana hairy roots for recombinant expression. We conducted a qualitative synthesis of 36 out of 387 publications initially selected. Following the PRISMA procedure, all papers were assessed for exclusion and inclusion criteria. Multiple points of root culture were explored, including transformation methods, root growth curve, external additives, and scale-up with bioreactors to determine which approaches performed best and what is still required to achieve a robust protocol. The information presented here may help researchers who want to work with hairy roots in their laboratories trace a successful path to high recombinant expression.

show abstract

The expression of the 14D9 catalytic antibody in suspended cells ofNicotiana tabacumcultures increased by the addition of protein stabilizers and by transference from erlenmeyer flasks to a 2‐L bioreactor

Cited by 7 publications

References 27 publications

High‐yield secretion of recombinant proteins expressed in tobacco cell culture with a designer glycopeptide tag: Process development

High‐yield secretion of recombinant proteins expressed in tobacco cell culture with a designer glycopeptide tag: Process development

Nicotiana hairy roots for recombinant protein expression, where to start? A systematic review

Nicotiana Hairy Roots for Recombinant Protein Expression, Where to Start? A systematic review

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