PMID- 28741464 OWN - NLM STAT- MEDLINE DCOM- 20181119 LR - 20191210 IS - 1875-5305 (Electronic) IS - 0929-8665 (Linking) VI - 24 IP - 11 DP - 2017 TI - Expression of Mastoparan B, a Venom Peptide, Via Escherichia coli C43 (DE3) Coupled with an Artificial Oil Body-Cyanogen Bromide Technology Platform. PG - 1021-1029 LID - 10.2174/0929866524666170724161900 [doi] AB - 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-beta-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 degrees C for 2 h after induction with 5 microM 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. CI - Copyright(c) Bentham Science Publishers; For any queries, please email at epub@benthamscience.org. FAU - Hsieh, Sheng-Kuo AU - Hsieh SK AD - School of Chinese Medicine, China Medical University, Taichung. Taiwan. FAU - Yu, Yu-Jen AU - Yu YJ AD - School of Chinese Medicine, China Medical University, Taichung. Taiwan. FAU - Tang, Nou-Ying AU - Tang NY AD - School of Chinese Medicine, China Medical University, Taichung. Taiwan. FAU - Lin, Jhao-Ren AU - Lin JR AD - Department of Pediatrics, Changhua Christian Hospital, Changhua. Taiwan. FAU - Jinn, Tzyy-Rong AU - Jinn TR AD - School of Chinese Medicine, China Medical University, Taichung. Taiwan. LA - eng PT - Journal Article PL - Netherlands TA - Protein Pept Lett JT - Protein and peptide letters JID - 9441434 RN - 0 (Anti-Bacterial Agents) RN - 0 (Intercellular Signaling Peptides and Proteins) RN - 0 (Peptides) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Venoms) RN - 137354-65-5 (mastoparan B) RN - 367-93-1 (Isopropyl Thiogalactoside) RN - OS382OHJ8P (Cyanogen Bromide) SB - IM MH - Amino Acid Sequence MH - Anti-Bacterial Agents/chemistry/pharmacology MH - Chromatography, High Pressure Liquid/methods MH - Cyanogen Bromide/*chemistry MH - Drug Delivery Systems/methods MH - Drug Liberation MH - Escherichia coli MH - Gene Expression MH - Humans MH - Intercellular Signaling Peptides and Proteins MH - Isopropyl Thiogalactoside/chemistry MH - Lipid Droplets/*chemistry MH - Particle Size MH - Peptides/*chemistry/*genetics/metabolism MH - Recombinant Fusion Proteins/chemistry/pharmacology MH - Venoms/*chemistry OTO - NOTNLM OT - Artificial oil bodies OT - Escherichia coli C43(DE3) OT - cyanogen bromide OT - mastoparan B OT - oleosin-fusion protein OT - venom peptide EDAT- 2017/07/26 06:00 MHDA- 2018/11/20 06:00 CRDT- 2017/07/26 06:00 PHST- 2017/05/09 00:00 [received] PHST- 2017/07/13 00:00 [revised] PHST- 2017/07/13 00:00 [accepted] PHST- 2017/07/26 06:00 [pubmed] PHST- 2018/11/20 06:00 [medline] PHST- 2017/07/26 06:00 [entrez] AID - PPL-EPUB-84956 [pii] AID - 10.2174/0929866524666170724161900 [doi] PST - ppublish SO - Protein Pept Lett. 2017;24(11):1021-1029. doi: 10.2174/0929866524666170724161900.