UBC Theses and Dissertations
Maximizing the expression of a cloned gene under the regulation of the metallothionein promoter Jervis, Eric J
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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