3.3 The enhanced expression of PCP vaccine proteins Oml1, Oml7,
and ApxⅡ
To investigate whether the expression intensity of the four bicistronic
T7 vectors will be affected by target gene sequences and assess their
efficacy in expressing other recombinant proteins, the four
best-performing T7 BCD systems were utilized to express three PCP
vaccine antigens Oml1, Oml7, and ApxII. Since the full-length ApxII
protein was observed to be expressed as an inclusion body in E.
coli (data not shown), the truncated ApxII fragment 5 (439-801 aa),
which has demonstrated good immunogenicity in previous studies, was
selected here for expression.[10,11] As shown in
SDS-PAGE, all three antigens were successfully expressed. Moreover, the
protein yield per OD600 of all four BCD vectors exceeded
that of the monocistronic control (Fig. 3A-3C ,). We further
calculated the relative protein yield per liter of cells for each
vector. The results in Fig. 3D-3F showed that the use of
pET28-HT5, pET28-HT8, pET28-HP11Y, and pET28-HT12Y increased the yield
of Oml1 by 38%, 29%, 29%, and 24%, the yield of Oml7 by 13%, 8%,
18%, and 17%, and the yield of ApxII by 24%, 18%, 22%, 17%,
respectively. These results suggested that all these four bicistronic T7
vectors have good compatibility and can enhance the expression of
various proteins. Based on these results, we selected the strongest
vector (pET28-HT5-Oml1, pET28-HP11Y-Oml7, and pET28-HT5-ApxII) for each
protein for subsequent protein expression experiments.
3.4 Optimization of cultivation temperature, induction
conditions, and medium components for the production of PCP vaccine
proteins in MWP
Apart from the expression element, cultivation temperature, induction
conditions, and medium components (such as carbon and nitrogen sources)
are also important factors that influence cell growth, cell wall
structure, and energy metabolism, thereby affecting protein
expression.[27–29] To determine the optimal
condition for the production of PCP vaccine proteins, we first
investigated the effect of temperature. As shown in Fig. 4A-4C ,
25°C was the most favorable temperature for protein expression among the
three temperatures tested, resulting in the highest expression level of
Oml1, Oml7, and ApxII. This beneficial effect of lower expression
temperature has also been observed in previous studies on the soluble
expression of recombinant proteins.[30,31]Therefore, for the subsequent expression of PCP vaccine proteins, the
cultivation temperature was set at 25°C.
We also optimized the IPTG concentration and pre-induction period for
PCP antigen expression. Increasing the IPTG concentration from 0 to 0.2
mM resulted in the appearance of Oml1 and Oml7 bands on SDS-PAGE, and
ApxII expression was first observed with 0.1 mM IPTG addition (data not
shown). No significant effect on cell growth was observed within the
IPTG concentration range tested (data not shown). Among the three
proteins tested, 0.2 mM IPTG was optimal, and the protein band did not
increase with further increases in IPTG concentration (Fig.
4D-F ). Therefore, 0.2 mM IPTG was used for PCP antigen production. The
effect of pre-induction period on protein production was shown inFig. 4G-4I , where a 2 h pre-induction period was found to be
optimal for the expression of Oml1, Oml7, and ApxII. lower protein
expression levels were observed when the pre-induction period was
shorter than 2 h. This may be because inducing protein expression during
the early growth phase increases the metabolic burden on the cell,
ultimately affecting cell growth and protein production. With the
further prolongation of pre-induction culture time, the production of
the three proteins decreased. Therefore, a 2-h pre-induction period was
chosen for subsequent experiments.
Additionally, the effects of different carbon and nitrogen sources on
PCP antigen production were also investigated. As shown in Fig.
5A-5C , among all carbon sources tested, glycerol had the most
significant positive effect on PCP antigen production. Compared to
glucose, the yield of Oml1, Oml7, and ApxII in glycerol increased by
30%, 15%, and 30%, respectively. The effect of nitrogen source on PCP
antigen expression was shown in Fig. 5D-5F . Unexpectedly, the
extra addition of nitrogen source urea,
(NH4)2SO4, and
NH4Cl to TBSB reduced cell growth and protein yield to
varying degrees. These findings suggested that the nitrogen source
present in the original TBSB is sufficient for both cell growth and
protein expression. Thus, in subsequent experiments, the nitrogen source
was added according to the original culture medium.