Abstract Royalisin is an antibacterial peptide previously isolated from the royal jelly of Apis mellifera. This peptide is toxic against a broad range of gram-positive bacteria, gram-negative bacteria and fungi. Royalisin contains 51 amino acid residues with 6 cysteine residues forming three intramolecular disulfide linkages, which may form a compact globular structure exhibiting high stability at low pH and high temperature. In this study, royalisin was overexpressed in Escherichia coli AD494 (DE3) as a recombinant protein fused with the C-terminus of oleosin by a linker polypeptide, intein S. Artificial oil bodies (AOBs) were reconstituted with triacylglycerol and phospholipid to obtain the insoluble recombinant protein. Royalisin was subsequently released from artificial oil bodies through self-splicing of intein induced by temperature alteration, and the recombinant royalisin was collected in the supernatant after centrifugation. Recombinant royalisin released from artificial oil bodies was folded to the optimal structure with its three intracellular disulfide bonds and exhibited high antibacterial activity. These results showed that the functional antibacterial peptide, royalisin, was successfully expressed and purified via the efficient AOB expression/purification system.
摘要 王漿抗菌蛋白 (royalisin) 為蜜蜂工蜂下咽頭腺 (hypopharyngeal glands) 分泌的抗菌胜肽，其由 51 個胺基酸所組成，大小為 5.5 KDa，其中有 6 個半胱胺酸 (cysteine) 組成三個分子內的雙硫鍵，而雙硫鍵可以提供一個緊密、穩定的球狀結構，因此在低 pH 和高溫處理下都可以保持結構及活性穩定。王漿抗菌蛋白在低濃度下 (1 µM) 具有抗菌殺菌的能力，主要可抗真菌、革蘭氏陽性菌及少數的革蘭氏陰性菌。在本研究中，將王漿抗菌蛋白成熟胜肽片段融合至油體膜蛋白 C 端，中間以蛋白質剪接子 (intein) 連接構築成為融合蛋白質 (fusion protein) 基因，並藉由大腸桿菌 AD494 大量表現。將重組後的融合蛋白與三酸甘油脂、磷脂質混合後，重組產生人造油體 (artificial oil body, AOB)；藉由溫度轉換，讓中間連接的 intein 可以自發性的誘導斷裂，使 AOB 上的王漿抗菌蛋白被釋放至水層。所釋放出的王漿抗菌蛋白經蛋白質電泳膠位移分析發現它具有分子內雙硫鍵並正確折疊，且有抗菌活性，顯示本研究成功地利用人造油體蛋白質純化系統表達及純化具高抗菌活性的王漿抗菌蛋白。
| Key words: royalisin, antimicrobial peptides, royal jelly, Apis mellifera, AOB expression/purification system | | 關鍵詞: 王漿抗菌蛋白、抗菌蛋白、蜂王漿、西洋蜂、人造油體。 |
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