Su Manman, Chang Weiqin, Zhang Kun, Cui Manhua, Wu Shuying, Xu Tianmin
Department of Obstetrics and Gynecology, The Second Hospital, Jilin University, Changchun, Jilin 130041, P.R. China.
Oncol Rep. 2016 Feb;35(2):1179-85. doi: 10.3892/or.2015.4448. Epub 2015 Nov 25.
Melittin is well known to possess cytolytic activity with wide-spectrum lytic properties and its potential use as an agent to treat several types of cancer has been tested. Due to the non-specific toxicity, melittin can impair not only cancer cells but also normal tissue. Thus, tumor-targeted toxins may be helpful for developing novel anticancer therapeutics. The urokinase-type plasminogen activator (uPA) plays a central role in tissue remodelling events occurring in normal physiology and in pathophysiology, including cancer invasion and metastasis. Heartening findings showed that uPA receptor is predominantly expressed on many types of cancer. Therefore, the amino-terminal fragment (ATF) of uPA which was able to identify and bond with cancer cells was used as the cell-targeting domain to make up tumor-targeted toxin in this study. In the present study, pPICZαC-ATF-melittin eukaryotic expression vector was successfully constructed. After transformed into P. pastoris and induced by methanol, rATF-mellitin was detected by SDS-PAGE and western blot analysis. After induction with methanol, the expression level of rATF-mellitin was 312 mg/l in 80-l fermentor. rATF‑mellitin was purified to >95% purity using SP Sepharose ion exchange chromatography and source™ 30 RPC with 67.2% recovery. Cell proliferation assay showed that rATF-melittin inhibited growth of SKOV3 cells and had no cytotoxicity effect on normal cells. For the first time, we established a stable and effective rATF-mellitin P. pastoris expression system to obtain a high level of expression of secreted rATF-mellitin which was purified by a highly efficient purification procedure.
蜂毒肽以具有细胞溶解活性和广泛的溶解特性而闻名，其作为治疗多种癌症药物的潜在用途已得到测试。由于非特异性毒性，蜂毒肽不仅会损害癌细胞，还会损害正常组织。因此，肿瘤靶向毒素可能有助于开发新型抗癌疗法。尿激酶型纤溶酶原激活剂（uPA）在正常生理和病理生理过程（包括癌症侵袭和转移）中发生的组织重塑事件中起核心作用。令人振奋的研究结果表明，uPA受体在多种类型的癌症中均有高表达。因此，本研究中能够识别并与癌细胞结合的uPA氨基末端片段（ATF）被用作细胞靶向结构域来构建肿瘤靶向毒素。在本研究中，成功构建了pPICZαC-ATF-蜂毒肽真核表达载体。将其转化到巴斯德毕赤酵母中并用甲醇诱导后，通过SDS-PAGE和蛋白质免疫印迹分析检测到了重组ATF-蜂毒肽。用甲醇诱导后，在80-L发酵罐中重组ATF-蜂毒肽的表达水平为312 mg/L。使用SP Sepharose离子交换色谱和source™ 30 RPC将重组ATF-蜂毒肽纯化至纯度>95%，回收率为67.2%。细胞增殖试验表明，重组ATF-蜂毒肽可抑制SKOV3细胞的生长，对正常细胞无细胞毒性作用。我们首次建立了一个稳定有效的重组ATF-蜂毒肽巴斯德毕赤酵母表达系统，以获得高水平表达的分泌型重组ATF-蜂毒肽，并通过高效纯化程序对其进行纯化。
