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.