Automatic inducer addition and harvesting of recombinant Escherichia coli cultures based on indirect on-line estimation of biomass concentration and specific growth rate
Nt. Eriksen et al., Automatic inducer addition and harvesting of recombinant Escherichia coli cultures based on indirect on-line estimation of biomass concentration and specific growth rate, BIOTECH BIO, 75(3), 2001, pp. 355-361
This article describes a novel bioreactor configuration for production opti
mization of recombinant proteins in Escherichia coli, Inducer addition and
harvesting are controlled on-line based on indirect estimation of biomass c
oncentration and specific growth rate from addition of NaOH to maintain con
stant pH. When either a predetermined biomass concentration is reached or t
he cultures have obtained, a constant specific growth rate inducer is intro
duced automatically, The induction period is ended by automatic harvesting
of the cultures either at a predetermined biomass concentration or when sub
strate (in this study glucose) is depleted, detected as an increase of pH,
or dissolved oxygen tension. During harvesting, metabolic activities are qu
enched within 3 min by cooling of the cell suspension. The system has been
used to optimize expression of glutathione S-transferase (GST) fusion prote
in of the ligand binding domain of mouse peroxisome proliferator-activated
receptor, GST-PPAR alpha LBD. Total yield of GST-PPAR alpha LBD was indepen
dent of the time of inducer addition as long as the length of induction per
iod corresponded to at least 0.25 cell divisions while the yield of soluble
GST-PPAR alpha LBD, the only active form, increased with the length of ind
uction period. Highest yields were obtained when the inducer was added at l
ow cell concentration as soon as constant specific growth rate was detected
, resulting in induction periods corresponding to 3.4 +/- 0.4 cell division
s. The specific growth rate remained almost constant for one cell division
after inducer addition, whereafter it decreased. No decrease of specific gr
owth rate was observed when inducer was added in the lag-phase, and no solu
ble protein was produced. These results suggest that solely soluble GST-PPA
R alpha LBD acts as a growth inhibitor and that GST-PPAR alpha LBD is expre
ssed predominantly as inclusion bodies immediately after inducer addition w
hereas the proportion expressed as soluble protein is increased after 1 h o
f induction, Compared to the procedures, which are generally used for prote
in expression in the laboratory, this system is less labor intensive, it au
tomatically provides recording of biomass concentration and specific growth
rate, and it allows direct comparisons between expression of different pro
teins and performance of different constructs since the induction period is
linked to growth. (C) 2001 John Wiley & Sons, Inc.