bioreactor studies of heterologous protein production by ...

4.3.3.4.Growth Rate Differences
For recombinant ce& both structurai instabiliity. segregational uistability? and growth rate
dinerence cm contribute to plasmid loss. Structural instability is ofkn caused by the
DNA's deletion, insertion, recombination, and other events, while segegational instability
is caused by a bias in partitionhg of plasmids between mother and daughter ceils during
ceII division (Primrose et al.. 198 1; Ryu and Lee, 1988). In addition, the plasmid-free celis
usuaiiy have a growth advantage over plasmid-bearing œlls in non-selective medium
because the presence of the foreign plasmid places additionai "stress and burden" on host
cells. The excess metaboüc load is due to the requirement to replicate and express the
foreign DNA (Seo and Baiiey, 1985; Bimaum and Baiïey, 1991). During ceU culture, the
plasmid-free ceUs tend to overwheirn the original population of p~asmid-bea~g ceils due
to their higher growth rate.
As shown in Table 4.3, there is negiigible dinerence between the growth rates of
plasmid-bearing and plasmid-free ceh at the dilution rates tested. which is in agreement
with the experimentai measurements of the maximum specific growth rates of the piasmici-
bearing and plasmid-kee ceUs for the current recombinant yeast saains (TMis et al., 1987).
No growth rate ciifferenas contribute to the apparent instability of the plasmids. This
ûnding agrees with the observation that the total ce4 concentration remained nearly
constant d d g the experiments (See Figure 4-17), even though the population of the
plasmid-bearing ceh continuously decreased (Figure 4.14). otherwise the total ceIl mas
would increase during the c u i process. ~ This result is also consistent with the fmding