Background: Mastoparan B (MPB) is a venom peptide isolated from Vespa basalis (black-bellied hornet), one of the dangerous vespine wasps found in Taiwan. MPB is a tetradecapeptide (LKLKSIVSWAKKVL), amphiphilic venom peptide, with a molecular mass of 1.6 kDa. MPB belongs to an evolutionarily conserved component of the innate immune response against microbes. In this study, we attempted to modify a reliable oleosin-based fusion expression strategy coupled with the artificial oil body (AOB)-cyanogen bromide (CNBr) platform to produce bioactive MPB.
Objectives: The aim of this study was to develop an artificial oil body (AOB)-cyanogen bromide (CNBr) platform to produce the bioactive form of mastoparan B (MPB), which in a manner identical to that of its native counterpart.
Methods: The plasmid pET30-His6-rOle(127M→L)-MPB was constructed, and then four different E. coli strains- BL21(DE3), BL21(DE3)pLysS, C41(DE3), and C43(DE3) were tested to identify the most suitable host for the pET30-His6-rOle(127M→L)-MPB fusion protein expression. We optimized the expression conditions by testing different growth temperatures, isopropyl-β-D-thiogalactoside (IPTG) concentrations, and post-induction collection times. Afterwards, the His6-rOle(127M→L)-MPB protein was purified by one-step nickel-chelated affinity chromatography (Ni2+-NTA) under denaturing conditions. The purified His6-rOle(127M→L)-MPB was selectively cleaved by thrombin protease to remove the His6-tag and the leader peptide from the N-terminus. Subsequently, rOle(127M→L)-MPB protein was constituted into AOB and incubated with CNBr for a cleavage reaction, which resulted in the release of the MPB from rOle(127M→L)-MPB protein via AOB. The purified MPB was identified by MALDI-MS and HPLC analysis, and its bioactivity was examined by antimicrobial testing.
Results: After a 2-h induction period, the E. coli C43(DE3) was found to be superior to BL21(DE3) and the other protease-deficient strains as an expression host. And, the optimal His6-rOle(127M→L)-MPB expression at 37°C for 2 h after induction with 5 µM IPTG. The purified MPB showed that a single major peak was detected by HPLC/UV detection with a retention time of 22.5 minutes, which was approximately 90% pure. The putative MPB, and over two-third of the peptide sequence was verified by the MALDI-MS analysis. Finally, the purified MPB was examined by a broth dilution-antimicrobial susceptibility test. These results indicated that the purified MPB was bioactive and very effective in anti-bacterial (E. coli J96) activity. Here, we successfully used the oleosin-based fusion expression strategy coupled with the artificial oil body (AOB)-cyanogen bromide (CNBr) platform to produce bioactive MPB peptide which, in a manner identical to that of its native counterpart.
Conclusion: In this study, the recombinant oleosin based fusion strategy coupled with AOB-CNBr purification platform open a new avenue for the production of active MPB and facilitate the studies and applications of the peptide in the future for medicinal applications such as hypotension and antibacterial effect.
Keywords: Artificial oil bodies, cyanogen bromide, Escherichia coli C43(DE3), mastoparan B, oleosin-fusion protein, venom peptide.
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