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Semicontinuous Bioreactor Production of Recombinant Butyrylcholinesterase in Transgenic Rice Cell Suspension Cultures.

Corbin JM, Hashimoto BI, Karuppanan K, Kyser ZR, Wu L, Roberts BA, Noe AR, Rodriguez RL, McDonald KA, Nandi S - Front Plant Sci (2016)

Bottom Line: The bioreactor was operated through two phases each of growth and expression.Cells successfully regrew during a 5-day growth phase.A combination of activity assays and Western blot analysis indicated production of an active and fully assembled tetramer of BChE.

View Article: PubMed Central - PubMed

Affiliation: Chemical Engineering and Materials Science, University of California, Davis Davis, CA, USA.

ABSTRACT
An active and tetrameric form of recombinant butyrylcholinesterase (BChE), a large and complex human enzyme, was produced via semicontinuous operation in a transgenic rice cell suspension culture. After transformation of rice callus and screening of transformants, the cultures were scaled up from culture flask to a lab scale bioreactor. The bioreactor was operated through two phases each of growth and expression. The cells were able to produce BChE during both expression phases, with a maximum yield of 1.6 mg BChE/L of culture during the second expression phase. Cells successfully regrew during a 5-day growth phase. A combination of activity assays and Western blot analysis indicated production of an active and fully assembled tetramer of BChE.

No MeSH data available.


Related in: MedlinePlus

Rice cell culture kinetics through two phases of cell growth and expression (as described in Section Materials and Methods) in a 5 L bioreactor. (A) Concentration of biomass in the culture in g of dry weight (DW) callus per L of total culture. (B) Oxygen uptake rate (OUR) of the culture in mmol O2 per L of culture per hour. (C) Concentration of glucose and sucrose found in the culture medium in g/L. (D) Total active cell-associated butyrylcholinesterase (BChE) in μg, normalized to g of fresh weight (FW) callus.
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Figure 3: Rice cell culture kinetics through two phases of cell growth and expression (as described in Section Materials and Methods) in a 5 L bioreactor. (A) Concentration of biomass in the culture in g of dry weight (DW) callus per L of total culture. (B) Oxygen uptake rate (OUR) of the culture in mmol O2 per L of culture per hour. (C) Concentration of glucose and sucrose found in the culture medium in g/L. (D) Total active cell-associated butyrylcholinesterase (BChE) in μg, normalized to g of fresh weight (FW) callus.

Mentions: The semicontinuous bioreactor culture was operated for 31 days and underwent two cycles each of growth and expression. Figure 3A shows the growth of the biomass over the duration of the culture. The first growth phase (Growth 1) had an initial biomass concentration of 0.21 ± 0.07 g DW/L of culture and lasted 16 days, while the second growth phase (Growth 2) had an initial biomass concentration of 2.37 ± 0.06 g DW/L and lasted 5 days. Growth 1 exhibited a long lag phase of 4 days followed by a long exponential phase of 12 days prior to induction, while Growth 2 had a lag of < 1 day and a 5 day exponential phase. This difference is likely due to the difference in the initial biomass concentration during each growth phase. During each expression phase, where NB+S was removed and replaced with NB−S, the biomass concentration immediately dropped as some of the biomass was pumped out of the reactor along with the spent medium. However, at the beginning of expression, the biomass concentration increased as the cells received fresh medium components such as nutrients and amino acids. The biomass concentration soon leveled off and began to drop as the cells starved from sugar deprivation, until a new growth phase was initiated. Table 1 summarizes the cell growth kinetics for both growth phases. The maximum specific growth rate (μmax) increased from 0.15 ± 0.01 day−1 during Growth 1 to 0.22 ± 0.01 day−1 during Growth 2, which correlated to a decrease in doubling time from 4.7 ± 0.3 to 3.2 ± 0.1 days.


Semicontinuous Bioreactor Production of Recombinant Butyrylcholinesterase in Transgenic Rice Cell Suspension Cultures.

Corbin JM, Hashimoto BI, Karuppanan K, Kyser ZR, Wu L, Roberts BA, Noe AR, Rodriguez RL, McDonald KA, Nandi S - Front Plant Sci (2016)

Rice cell culture kinetics through two phases of cell growth and expression (as described in Section Materials and Methods) in a 5 L bioreactor. (A) Concentration of biomass in the culture in g of dry weight (DW) callus per L of total culture. (B) Oxygen uptake rate (OUR) of the culture in mmol O2 per L of culture per hour. (C) Concentration of glucose and sucrose found in the culture medium in g/L. (D) Total active cell-associated butyrylcholinesterase (BChE) in μg, normalized to g of fresh weight (FW) callus.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4814504&req=5

Figure 3: Rice cell culture kinetics through two phases of cell growth and expression (as described in Section Materials and Methods) in a 5 L bioreactor. (A) Concentration of biomass in the culture in g of dry weight (DW) callus per L of total culture. (B) Oxygen uptake rate (OUR) of the culture in mmol O2 per L of culture per hour. (C) Concentration of glucose and sucrose found in the culture medium in g/L. (D) Total active cell-associated butyrylcholinesterase (BChE) in μg, normalized to g of fresh weight (FW) callus.
Mentions: The semicontinuous bioreactor culture was operated for 31 days and underwent two cycles each of growth and expression. Figure 3A shows the growth of the biomass over the duration of the culture. The first growth phase (Growth 1) had an initial biomass concentration of 0.21 ± 0.07 g DW/L of culture and lasted 16 days, while the second growth phase (Growth 2) had an initial biomass concentration of 2.37 ± 0.06 g DW/L and lasted 5 days. Growth 1 exhibited a long lag phase of 4 days followed by a long exponential phase of 12 days prior to induction, while Growth 2 had a lag of < 1 day and a 5 day exponential phase. This difference is likely due to the difference in the initial biomass concentration during each growth phase. During each expression phase, where NB+S was removed and replaced with NB−S, the biomass concentration immediately dropped as some of the biomass was pumped out of the reactor along with the spent medium. However, at the beginning of expression, the biomass concentration increased as the cells received fresh medium components such as nutrients and amino acids. The biomass concentration soon leveled off and began to drop as the cells starved from sugar deprivation, until a new growth phase was initiated. Table 1 summarizes the cell growth kinetics for both growth phases. The maximum specific growth rate (μmax) increased from 0.15 ± 0.01 day−1 during Growth 1 to 0.22 ± 0.01 day−1 during Growth 2, which correlated to a decrease in doubling time from 4.7 ± 0.3 to 3.2 ± 0.1 days.

Bottom Line: The bioreactor was operated through two phases each of growth and expression.Cells successfully regrew during a 5-day growth phase.A combination of activity assays and Western blot analysis indicated production of an active and fully assembled tetramer of BChE.

View Article: PubMed Central - PubMed

Affiliation: Chemical Engineering and Materials Science, University of California, Davis Davis, CA, USA.

ABSTRACT
An active and tetrameric form of recombinant butyrylcholinesterase (BChE), a large and complex human enzyme, was produced via semicontinuous operation in a transgenic rice cell suspension culture. After transformation of rice callus and screening of transformants, the cultures were scaled up from culture flask to a lab scale bioreactor. The bioreactor was operated through two phases each of growth and expression. The cells were able to produce BChE during both expression phases, with a maximum yield of 1.6 mg BChE/L of culture during the second expression phase. Cells successfully regrew during a 5-day growth phase. A combination of activity assays and Western blot analysis indicated production of an active and fully assembled tetramer of BChE.

No MeSH data available.


Related in: MedlinePlus