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Maximizing the expression of a cloned gene under the regulation of the metallothionein promoter Jervis, Eric J
Abstract
A stably transformed BHK cell line, engineered to produce a human transferrin half-molecule under the control of a mouse metallothionein promoter, was used as a model system to develop strategies to maximize recombinant protein productivity in mammalian cell culture. The metallothionein promoter is inducible and promotes high levels of expression. Fully induced cells produced up to 0.7 pg transferrin/cell hr, a 4-fold increase in transferrin production over uninduced levels. Cell growth was inhibited at cadmium dosages above 1.0 fmole/cell; prolonged exposure at this dosage is cytotoxic. The metal dose-dependence of induction and lethal effects at high metal dosages motivated the development of special strategies to maximize gene expression. Dosing regimes which maintained cell associated cadmium concentrations below 0.25 fmole/cell, ensured cell growth and high cell specific productivities which maximized final product titers. For routine batch culture initial inducer loadings of 10 fmole/cell for zinc and 1.0 fmole/cell for cadmium gave near-maximum transferrin production levels. For extended culture, repeated small doses of between 0.25 fmole/cell and 0.45 fmole/cell, based on initial cell counts, every 48 hours maximized transferrin synthesis with this cell line. A novel compartmental model with 3 inducer pools was developed which quantitatively reproduced many of the experimental results. Dose-dependent compartmental transfer functions were modeled and kinetic parameters estimated for transferrin synthesis and secretion. Several batch and fedbatch inducer dosing strategies were simulated to identify protocols which maximized gene expression. Sensitivity analysis of the model's parameters predicted that transferrin production rates are less sensitive to variation in cell specific cadmium uptake rates at lower inducer loadings.
Item Metadata
Title |
Maximizing the expression of a cloned gene under the regulation of the metallothionein promoter
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Creator | |
Publisher |
University of British Columbia
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Date Issued |
1992
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Description |
A stably transformed BHK cell line, engineered to produce a human transferrin half-molecule
under the control of a mouse metallothionein promoter, was used as a model system to
develop strategies to maximize recombinant protein productivity in mammalian cell culture.
The metallothionein promoter is inducible and promotes high levels of expression. Fully
induced cells produced up to 0.7 pg transferrin/cell hr, a 4-fold increase in transferrin
production over uninduced levels. Cell growth was inhibited at cadmium dosages above 1.0
fmole/cell; prolonged exposure at this dosage is cytotoxic. The metal dose-dependence of
induction and lethal effects at high metal dosages motivated the development of special
strategies to maximize gene expression. Dosing regimes which maintained cell associated
cadmium concentrations below 0.25 fmole/cell, ensured cell growth and high cell specific
productivities which maximized final product titers. For routine batch culture initial inducer
loadings of 10 fmole/cell for zinc and 1.0 fmole/cell for cadmium gave near-maximum
transferrin production levels. For extended culture, repeated small doses of between 0.25
fmole/cell and 0.45 fmole/cell, based on initial cell counts, every 48 hours maximized
transferrin synthesis with this cell line. A novel compartmental model with 3 inducer pools was
developed which quantitatively reproduced many of the experimental results. Dose-dependent
compartmental transfer functions were modeled and kinetic parameters estimated for
transferrin synthesis and secretion. Several batch and fedbatch inducer dosing strategies were
simulated to identify protocols which maximized gene expression. Sensitivity analysis of the
model's parameters predicted that transferrin production rates are less sensitive to variation in
cell specific cadmium uptake rates at lower inducer loadings.
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Extent |
4964548 bytes
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Genre | |
Type | |
File Format |
application/pdf
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Language |
eng
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Date Available |
2008-12-17
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Provider |
Vancouver : University of British Columbia Library
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Rights |
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
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DOI |
10.14288/1.0058515
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URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
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Graduation Date |
1992-11
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Campus | |
Scholarly Level |
Graduate
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Aggregated Source Repository |
DSpace
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Item Media
Item Citations and Data
Rights
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.