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.