Bioreactor cultivations
For the pre-culture, 500 mL of semi-defined medium were inoculated with
a frozen stock in a 2500 mL High Yield shake flask and incubated for 16
h at 37°C and 230 rpm in an Infors HR Multitron incubator (Infors,
Bottmingen, Switzerland).
The plasmid free X-press strain was cultivated in a stainless steel
bioreactor with a working volume of 10 L (Biostat Cplus, Sartorius,
Göttingen, Germany). The batch volume was 5 L. The culture broth was
supplied with a mixture of air and pure oxygen at 10 L/min and stirred
constantly at 1200 rpm. Dissolved oxygen (DO) was monitored using a
fluorescence electrode (Visiferm DO120, Hamilton, Reno, NV, USA) and
kept above 35% by adjusting the amount of added pure oxygen. pH was
monitored with an Easyferm electrode (Hamilton) and kept constant at
7.00 via addition of NH4OH (12.5%). The temperature was
controlled with the built-in heat jacket and kept at 37°C, except during
induction (described below).
The recombinant protein production processes were carried out in a
DASGIP parallel reactor system (Eppendorf, Hamburg, Germany) with four
vessels containing 2 L working volume, aerated at 2 L/min. The batch
volume was 1 L. Gas mixing and control of DO, pH and temperature (via
heat blanket and cooling finger) were done analogously to the
cultivations in the stainless steel bioreactor described above.
The batch was started by inoculating minimal media (90% of the batch
volume) with the preculture (10% of the batch volume). Once glucose was
depleted (detected by a DO spike), substrate was fed to reach a cell dry
weight concentration of 50 g/L and 30 g/L in the growth repression and
recombinant protein production processes, respectively. Subsequently,
expression of Protein A or VHH was induced by addition of 0.5 mM or 0.25
mM IPTG, respectively. Additionally, Gp2 expression in the X-press
strain was induced by adding 100 mM L-arabinose.