Introduction
Actinobacillus pleuropneumonia (A. pleuropneumonia ) is the causative agent of porcine contagious pleuropneumonia (PCP), a highly contagious and usually fatal disease of pigs.[1,2]It has caused considerable economic losses to the global pig-rearing industry and is also one of the five internationally recognized diseases that endanger the pig industry. Although antibiotics can prevent PCP outbreaks, the abuse of antibiotics has led to drug resistance of pathogenic bacteria, and the policy of restrictive antibiotics in livestock and poultry feeding products in many countries drives up the demand for PCP vaccines.[3,4] Currently, the PCP vaccines mainly used are whole cell inactivated vaccines of several pathogenic serotypes, with poor-cross protection.[5] Recently, subunit vaccine derived from the immune-protective antigens of A. pleuropneumoniae , such as Apx toxins and outer membrane lipoprotein (Oml), common to many serotypes of A. pleuropneumonia strains, have demonstrated strong cross-immune effect and good prospects for preventing PCP.[5,6] Among the numerous Apx toxins, ApxII is the most promising vaccine candidate, as all 15 serotypes of A. pleuropneumoniae except serotype 10 express it.[6,7] So far, laboratories and industry have successfully cloned and expressed Oml and ApxII in E. coli , yeast, Corynebacterium glutamicum (C. glutamicum ), etc.[8–12] However, E. coli is still the most widely used expression system for the production of PCP subunit vaccine proteins, and the current recombinant expression in E. coli exists some drawbacks that need to be further improved. For example, protein yields are low, especially for ApxII.[10] Most recombinant ApxII existed in an inactive inclusion body form, resulting in complicated downstream work and substantial loss of the target protein. Therefore, the efficient expression of PCP subunit vaccine proteins in E. coli remains a huge challenge.
To improve the production of recombinant proteins in E. coli , several strategies have been adopted by researchers, including developing strong promoters and ribosome binding sites (RBSs), modifying host cells, and optimizing fermentation conditions, etc.[13–16] Among them, the operation of promoters is considered the most effective way, as promoters confer direct control of the mRNA abundance for protein translation and can determine nearly 80% of the protein expression level. To date, promoters from different origins, such as bacteria (Lac, tac, trp, and araBAD) and bacteriophage (T7, T5, and SP6), have been developed and applied in E. coli .[14] Due to its outstanding performance, pET expression vector based on the T7 promoter is still the most widely used and popular commercial expression system.[14,17] According to statistics, more than 90% of the 2003 PDB proteins were produced using the T7 promoter.[17]
Despite the effectiveness of strong promoters in enhancing gene expression, other genetic elements, such as unfavorable mRNA secondary structures in the translation initiation region (TIR), can also limit the expression level of target genes.[18]Optimizing the TIR region for each target gene can solve this problem but cumbersome and time-consuming.[19]Alternatively, the bicistronic design (BCD) can be utilized to overcome this issue.[20] In the BCD expression cassette, a short coding sequence (CDS) is inserted as the fore-cistron upstream of the target gene, the intrinsic helicase activity of ribosome recruited by the translation of fore-cistron can disrupt the mRNA structure around the target gene TIR, thus faciliting its translation initiation.[20,21] Since the fore-cistronic peptide has almost no functional purpose in BCD, the fore-cistron can be flexibly modified and replaced as an independent genetic element. A well-translated fore-cistron can be introduced as an “enhancer” to further improve the expression level of the target protein through translation coupling. For example, in our previous study, the expression of seven protein models was greatly enhanced in C. glutamicum by using bicistronic Ptac systems containing well-performed fore-cistron sequences.[19] Although BCD has proven effective in many prokaryotic expression systems, given the complexity of the expression frame, constructing a new bicistronic system to enhance recombinant protein expression is not a straightforward task. Sequences that allow efficient ribosome translation are not always suitable as fore-cistrons.[19,22,23] Therefore, to ensure that the newly constructed BCD system effectively enhances protein production, optimizing the genetic elements, especially the fore-cistron sequence, is necessary.
In this study, to achieve the hyper-production of PCP subunit vaccine proteins in E. coli , we built the bicistronic T7 expression system. We evaluated the effect of a series of fore-cistron sequences on BCD expression intensity and selected the top four strongest BCD vectors for the expression of three PCP vaccine proteins, Oml1, Oml7, and ApxII. Among them, Oml1 and Oml7 are the outer membrane lipoprotein of A. pleuropneumoniae serotypes 1 and 7 frequently appeared in China. Optimal culture conditions, induction conditions, and medium composition were also established to further improve protein yields. Finally, fed-batch cultivation in a 5 L bioreactor resulted in unprecedented high yields of Oml1 (2.43 g/L), Oml7 (2.59 g/L), and ApxII (1.21 g/L), and these recombinant antigens exhibited good immune protective efficacy in mouse models.