4. Discussion
To date, various expression systems have been developed for the
production of recombinant proteins, including mammalian cells, insects,
plants, yeast, and bacteria.[13,33,34] Among them,
the Gram-negative E. coli has long been the “workhorse” of
recombinant proteins due to its easy manipulation, rapid growth, clear
genetic background, and high-yielding recombinant protein expression,
etc.[35,36] For the recombinant expression inE. coli , the expression vector is a key component to determine
the protein expression level.[14] Generally, to
obtain satisfactory protein yields, expression vectors containing strong
promoters are used preferentially. Although numerous strong promoters
have been developed for the production of recombinant proteins over the
past decades,[14] the T7 promoter and its derived
pET expression vector are still the most widely used and popular
commercial expression systems in E. coli . In this study, the
bicistronic strategy was applied to the T7-based pET28 expression system
to further enhance its expression intensity.
Bicistronic expression design, a structure derived from the polycistron
in archaea and bacteria, has several advantages over conventional
monocistronic expression for recombinant protein production. Due to the
prior translation of the fore-cistron, the mRNA secondary structure
around the target gene’s translational initiation region (TIR) can be
reduced.[20] Moreover, there is translation
coupling in bicistronic expression, where the upstream fore-cistron can
significantly impact the expression of the downstream target
gene.[20,37] This means that a well-translated
upstream fore-cistron sequence can be introduced as an “enhancer” to
further improve protein yield. This strategy has been applied to improve
the expression intensity of tac promoter in C.
glutamicum ,[19] in this study, we further applied
it to the T7 expression system. To ensure the successful enhancement of
recombinant expression, a series of bicistronic T7 systems containing
various fore-cistron sequences were constructed and evaluated. Four of
the most effective bicistronic T7 systems were then used to address the
low expression status of the PCP subunit vaccine in E. coli . As
shown in Figure 3 , all four BCD vectors exhibited good
compatibility and an enhancement effect on the expression of three
antigen proteins (Oml1, Oml7, and ApxII). This observed good
compatibility can be attributed to the reduction of unfavorable mRNA
structures by the translation of fore-cistron, which decreases the
impact of the target gene sequence on gene expression
level.[20,38] After several rounds of optimization
in MWP, the production of Oml1, Oml7, and ApxII in the T7 BCD system
gradually increased, and achieved the highest yields reported so far,
reaching 2 .43 g/L, 2.59 g/L, and 1.21 g/L in a 5-L bioreactor,
respectively (Fig. 3 ). Despite these satisfactory results,
there is still much work that can be done. For example, the output of
the T7 BCD system still has great room for improvement, the expression
intensity of the T7 BCD system can be further improved by optimizing
other genetic elements, such as SD2 and the length of fore-cistron.
Although the three recombinant antigens produced here induced
well-protective immunity against A. pleuropneumoniae , the immune
protective effect of conjugate antigens Oml1/ApxII, Oml7/ApxII, and
Oml1/Oml7/ApxII remains to be evaluated, as previous studies have
reported an improved survival rate through combined immunization with
Apx toxin and Oml.[4] Additionally, the
application of the T7 BCD systems should be extended to the expression
of other proteins to further evaluate its expression performance.
In conclusion, this study successfully established a bicistronic T7
expression system for enhancing recombinant protein production inE. coli , which led to hyper-production of PCP subunit vaccine
proteins Oml1, Oml7, and ApxII. The unprecedented yields of these
proteins were achieved through a combination of the T7 BCD system and
fermentation condition optimization in MWP, with final yields of 2.43
g/L for Oml1, 2.59 g/L for Oml7, and 1.21 g/L for ApxII in a 5-L
bioreactor. This study provided a powerful protein expression system for
the enhanced production of recombinant proteins or overexpression of key
enzymes in E. coli . In addition, it also promoted the development
of subunit vaccines against PCP and provided an excellent strain for the
production of PCP subunit vaccine proteins.