Bílikova Katarina, Huang Sheng-Chang, Lin I-Ping, Šimuth Jozef, Peng Chi-Chung
Institute of Forest Ecology, Slovak Academy of Sciences, Zvolen, Detasched workplace Department of Molecular Apidology, Bratislava, Slovakia.
Department of Biotechnology, National Formosa University, Yunlin, Taiwan.
Peptides. 2015 Jun;68:190-6. doi: 10.1016/j.peptides.2015.03.001. Epub 2015 Mar 14.
Royalisin is a 5.5-kDa antibacterial peptide isolated from the royal jelly of the honeybee (Apis mellifera). The antimicrobial activity of royalisin against fungi, Gram-positive and Gram-negative bacteria has been revealed. Compared with another insect antibacterial peptide, there is an extra stretch of 11 amino acid residues at the C-terminus of royalisin. In this study, a recombinant shortened form of royalisin named as royalisin-D, was constructed without the 11 amino acid residues at the C-terminal of royalisin and linked to the C-terminal of oleosin by an inteinS fragment. The recombinant protein was overexpressed in Escherichia coli, purified by artificial oil body system and subsequently released through self-splicing of inteinS induced by the changes of temperature. The antibacterial activity of royalisin-D was compared with royalisin via minimal inhibitory concentration (MIC) assay, minimal bactericidal concentration (MBC) assay, microbial adhesion to solvents (MATS) methods, and cell membrane permeability. Furthermore, the recombinant royalisin and royalisin-D have also been treated with the reducing agent of disulfide bonds, dithiothreitol (DTT), to investigate the importance of the intra-disulfide bond in royalisin. In our results, royalisin-D exhibited similar antimicrobial activity to royalisin. Royalisin and royalisin D lost their antimicrobial activities when the intra-disulfide bonds were reduced by DDT. The intra-disulfide bond plays a more important role than the extra stretch of 11 amino acid residues at the C-terminus of royalisin in terms of the antimicrobial properties of the native royalisin.
王浆抗菌肽是一种从蜜蜂（西方蜜蜂）蜂王浆中分离出的5.5千道尔顿抗菌肽。已揭示王浆抗菌肽对真菌、革兰氏阳性菌和革兰氏阴性菌的抗菌活性。与另一种昆虫抗菌肽相比，王浆抗菌肽的C末端有一段额外的11个氨基酸残基。在本研究中，构建了一种重组的缩短形式的王浆抗菌肽，命名为王浆抗菌肽-D，它没有王浆抗菌肽C末端的11个氨基酸残基，并通过内含肽S片段连接到油质蛋白的C末端。该重组蛋白在大肠杆菌中过表达，通过人工油体系统纯化，随后通过温度变化诱导的内含肽S的自剪接而释放。通过最小抑菌浓度（MIC）测定、最小杀菌浓度（MBC）测定、微生物对溶剂的粘附（MATS）方法和细胞膜通透性，比较了王浆抗菌肽-D与王浆抗菌肽的抗菌活性。此外，还使用二硫键还原剂二硫苏糖醇（DTT）处理重组王浆抗菌肽和王浆抗菌肽-D，以研究王浆抗菌肽中链内二硫键的重要性。在我们的结果中，王浆抗菌肽-D表现出与王浆抗菌肽相似的抗菌活性。当链内二硫键被DDT还原时，王浆抗菌肽和王浆抗菌肽D失去了它们的抗菌活性。就天然王浆抗菌肽的抗菌特性而言，链内二硫键比王浆抗菌肽C末端额外的11个氨基酸残基发挥更重要的作用。
