3.4 Analysis of key factors for controlling autolysis
To investigate whether the changes in the transcription level of T7 RNAP
caused the difference in protein expression, the transcription levels of
4 promoters were measured by qRT-PCR. As shown in Figure 5A, the
strength of the 4 promoters were lac-1A
<lac-1G<lacUV5<lacUV5-1A. The
transcription level of T7 RNAP from Plac-1G was only
16.4% that of PlacUV5. Consequently, the former
maintained GDH expression at a slightly lower level, which might be the
key reason for suppression of PCD. In a previous study, the expression
capacity of T7 RNAP within a wide range was realized by designing RBS,
which precisely regulate the expression of recombinant proteins (Liang
et al., 2018). However, compared to Plac-1A, the
relative transcription level of Plac-1G was increased by
2.05 times, which resulted in faster GDH accumulation than in BL21
(DE3-lac1A), which finally led to the highest performance.
Interestingly, the transcription level of the lacUV5-1A promoter showed
a 2.68 folds increase, indicating that a strong promoter is not
conducive to the expression of GDH and may be the key reason for PCD.
In addition to controlling the expression level of T7 RNAP, we
investigated if it could also be prevented by modifying the copy number
of the plasmid carrying the target gene. High copy number plasmid for
protein production puts extra metabolic load on host cells, resulting
inhibition of cell growth and plasmid instability (Bentley et al.,
2009). In a previous study, sequence homology between CoIE1 RNA I/ RNA
II and tRNAs was abolished to keep the plasmid copy number constant,
then metabolic activity can be prolonged (Grabherr et al., 2002). Based
on this, the replicon of the pET plasmid (CoIE1) with 15 copies was
replaced by the RK2 replicon with 5 copies. Then, the BL21
(DE3-lac1G)-R2K and BL21 (DE3)-R2K strains with combinations of “strong
+ low copies” and “weak + low copies” were obtained, respectively. As
shown in Fig. 5B, although the GDH activity of BL21 (DE3)-R2K was higher
than that of BL21 (DE3) -colE1, it was only 51.62% that of the BL21
(DE3-lac1G)-colE1. The “weak + low copies” group exhibited much lower
enzyme activity than the other groups. Overall, controlling the
expression level of T7 RNAP is more effective than controlling the copy
number of the plasmid, implying that T7 RNAP plays the role of the lysis
“switch” in the pET system for recombinant protein overexpression
(Fig. 5C).