Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing, 210046, China.
Appl Microbiol Biotechnol. 2013 Apr;97(7):2877-84. doi: 10.1007/s00253-012-4541-y. Epub 2012 Nov 13.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), as an anticancer protein with tumor-selective apoptotic activity, has been examined for use in clinical application. Melittin, an antibacterial peptide isolated from the bee Apis mellifera, has shown strong cytotoxicity to both tumor and normal cells. To ameliorate the cytotoxicity of melittin on cells and enhance the activity of TRAIL on cancer cells, we constructed a novel fusion protein, sTRAIL-melittin, containing a small ubiquitin-related modifier (SUMO) tag and expressed this fusion protein in Escherichia coli. Data showed that expression of the soluble fusion protein with the SUMO tag was approximately 85% of total target protein which was much higher than that without the SUMO tag (approximately 10%); sTRAIL-melittin was easily purified using Ni-NTA affinity chromatography and the tag was removed easily using SUMO-specific protease. To assay anticancer activity and side effects, methyl thiazolyl tetrazolium, hemolytic, and apoptosis assays were employed. Results demonstrated that sTRAIL-melittin had cytotoxic and apoptotic activity in K562 leukemia cells and HepG2 liver carcinoma cells, while it had only a minimal effect on erythrocytes and normal HEK293 cells. This indicates that the cytotoxicity of sTRAIL-melittin in normal cells was low and the anticancer activity of the fusion protein in tumor cells was significantly enhanced compared with sTRAIL (P<0.01). Furthermore, we found that sTRAIL-melittin also showed antibacterial activity to Staphylococcus aureus due to the presence of the melittin domain. Therefore, TRAIL fused with an antibacterial peptide may be a promising novel TRAIL-based anticancer treatment strategy.
肿瘤坏死因子相关凋亡诱导配体（TRAIL）作为一种具有肿瘤选择性凋亡活性的抗癌蛋白，已被研究用于临床应用。蜂毒肽是从蜜蜂 Apis mellifera 中分离出的一种抗菌肽，对肿瘤细胞和正常细胞均显示出很强的细胞毒性。为了减轻蜂毒肽对细胞的细胞毒性并增强 TRAIL 对癌细胞的活性，我们构建了一种新型融合蛋白 sTRAIL-melittin，该蛋白含有一个小泛素相关修饰物（SUMO）标签，并在大肠杆菌中表达了这种融合蛋白。数据表明，表达带有 SUMO 标签的可溶性融合蛋白的总靶蛋白约为 85%，远高于不带有 SUMO 标签的蛋白（约为 10%）；sTRAIL-melittin 可通过 Ni-NTA 亲和层析轻松纯化，并且标签可通过 SUMO 特异性蛋白酶轻松去除。为了检测抗癌活性和副作用，采用甲基噻唑基四唑、溶血和凋亡测定法。结果表明，sTRAIL-melittin 在 K562 白血病细胞和 HepG2 肝癌细胞中具有细胞毒性和凋亡活性，而对红细胞和正常 HEK293 细胞的影响很小。这表明 sTRAIL-melittin 在正常细胞中的细胞毒性较低，并且与 sTRAIL 相比，融合蛋白在肿瘤细胞中的抗癌活性显著增强（P<0.01）。此外，我们发现由于存在蜂毒肽结构域，sTRAIL-melittin 对金黄色葡萄球菌也具有抗菌活性。因此，与抗菌肽融合的 TRAIL 可能是一种有前途的新型基于 TRAIL 的抗癌治疗策略。
