STRATEGIES FOR MAXIMIZING METALLOTHIONEIN PROMOTER REGULATED RECOMBINANT PROTEIN-PRODUCTION IN MAMMALIAN-CELL CULTURES

A stably transformed BHK cell line, engineered to produce a human tran sferrin half-molecule under the control of a mouse metallothionein (MT ) promoter, was used as a model system to develop strategies to increa se inducible recombinant protein production. Gene expression regulated by the MT promoter is induced by heavy metals (e.g. Zn+2 or Cd+2) in a dose dependent fashion. However, at high concentrations these metals are toxic to cells. Culture protocols which balance these counteracti ve effects are needed to maximize transferrin production. Fully induce d cells produced up to 0.7 pg transferrin/cell . h, a 3-fold increase in production over uninduced levels. Cell growth was inhibited at Cd+2 dosages above 1 fmol/cell; prolinged exposure at this dosage was cyto toxic. Cell specific transferrin productivities decreased within 48 h following induction with Cd+2 although cell-associated Cd+2 levels rem ain high. Further addition of Cd+2 to cultures restored cell specific transferrin production rates. This suggests that cell associated Cd+2 is sequestered into a form which does not stimulate the MT promoter. C d+2 dosing regimes which maintained cell associated Cd+2 concentration s between 0.2 and 0.35 fmol/cell ensured cell growth and high cell spe cific productivities which maximized final product titers. For routine batch culture, initial Cd+2 loadings of 0.8 fmol/cell gave near-maxim um transferrin production levels. For extended culture, repeated small doses of 0.5 fmol/cell every 24 to 48 h maximized transferrin synthes is with this cell line.