Maximizing the expression of a cloned gene under the regulation of the metallothionein promoter
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...
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Format | Dissertation |
Language | English |
Published |
17.12.2008
1992 01.11.1992 |
Online Access | Get more information |
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Summary: | 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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