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.