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).