E. Jervis et Dg. Kilburn, STRATEGIES FOR MAXIMIZING METALLOTHIONEIN PROMOTER REGULATED RECOMBINANT PROTEIN-PRODUCTION IN MAMMALIAN-CELL CULTURES, Cytotechnology, 21(3), 1996, pp. 217-223
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.