3.4 Optimization of production conditions for POI
A convenient quantification method for the expressed POI is essential
for the optimization of bioproduction conditions. The relationship
between FL-DT fluorescence intensity and the concentration of
N2C was then investigated by incubating 0.01-1.00 equivalent of N2C withFL-DT for 30 min (Figure S7B ). The presence of N2C
triggered the release of fluorescence, which is also observed under a
handheld UV lamp (Figure 4C ). The changes in fluorescence
intensity were in a linear relationship with the concentrations of the
added N2C (Figure S7C ). The limit of detection was determined
to be 2 nM. The concentrations of N2C expressed in E. coli under
variable production conditions, including the concentration of inducer
isopropyl-β -D-thiogalactopyranoside (IPTG), induction time, and
incubation temperature, were then quantified with FL-DT to
determine the optimal parameters. Initially, three IPTG concentrations
(0.1 mM, 0.5 mM, and 1.0 mM) were investigated for their impacts on
protein expression. Each test group was subjected to 3 parallel
experiments at 30 °C with an incubation time of 20 h. The fluorescence
intensity was recorded and converted to protein concentration (method
described in supplementary materials). The result reveals that the
concentration of inducer IPTG barely affects the N2C production
efficiency (Figure S7D ). By fixing the inducer concertation at
0.5 mM, the effect of different induction temperatures (20 °C, 25°C,
30°C, 37°C) on protein expression at 20 h was explored, showing an
optimal induction temperature of 30°C (Figure 4D ). The contents
of N2C expression after 8 h, 12 h, 16 h, 20 h, and 24 h were then
compared under 30°C with the inducer concertation of 0.5 mM(Figure 4E ). The effect of induction temperature and culture
time have a remarkable influence on the expression of recombinant
protein, and the optimized condition for N2C protein production of
~0.48 mg/mL is to incubate for 20 h at 30 °C.