Preparation and characterization of a novel variant of human tumor necrosis factor-related apoptosis-inducing ligand from the rhesus monkey, Macaca mulatta
Preparation and characterization of a novel variant of human tumor necrosis factor-related apoptosis-inducing ligand from the rhesus monkey, Macaca mulatta
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DOI：
10.1007/s00253-015-7143-7
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发表时间：
2016-04-01
影响因子：
5
通讯作者：
Lu, Xiaofeng
中科院分区：
文献类型：
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作者：
Jia, Dianlong;Yang, Hao;Lu, Xiaofeng
人肿瘤坏死因子相关凋亡诱导配体（hTRAIL）及其变异体是一种很有吸引力的抗肿瘤候选药物。预测的氨基酸序列的功能性细胞外结构域的猕猴TRAIL（mmTRAIL）被发现不同的hTRAIL在四个位置。在本研究中，编码mmTRAIL的基因被克隆并在大肠杆菌中重组表达，产量约为20 - 30 mg/L，这是hTRAIL的两倍。SDS-PAGE显示变性后的mmTRAIL与hTRAIL分子量相近。然而，尺寸排阻色谱和动态光散射（DLS）分析表明，天然mmTRAIL的分子大小小于天然hTRAIL的分子大小。将这些蛋白质的溶液从室温冷却至0 A ° C诱导hTRAIL而不是mmTRAIL的大量沉淀，表明mmTRAIL在低温下比hTRAIL更可溶。此外，mmTRAIL比hTRAIL对N-溴代琥珀酰亚胺（NBS）诱导的沉淀更具抗性。尽管mmTRAIL和hTRAIL对人死亡受体显示出相当的纳摩尔亲和力，但mmTRAIL-受体复合物的解离速率比hTRAIL-受体复合物的解离速率慢，表明mmTRAIL-受体复合物更稳定。此外，mmTRAIL在人肿瘤细胞中诱导半胱天冬酶依赖性凋亡，其IC50比hTRAIL低2 - 3倍。然而，体内评价表明，mmTRAIL或hTRAIL在携带COLO 205异种移植物的小鼠中导致相似水平的肿瘤抑制。尽管如此，其更好的溶解性的优势应该促进mmTRAIL在癌症生物治疗中的生产和进一步使用。
Human tumor necrosis factor-related apoptosis-inducing ligand (hTRAIL) and its variants are attractive antitumor drug candidates. The predicted amino acid sequence of the functional extracellular domain of Macaca mulatta TRAIL (mmTRAIL) was found to differ from that of hTRAIL at four positions. In this study, the gene encoding mmTRAIL was cloned and recombinantly expressed in Escherichia coli at a yield of approximately 20-30 mg/L, which was two times higher than that of hTRAIL. SDS-PAGE showed that denatured mmTRAIL and hTRAIL had similar molecular weights. However, size-exclusion chromatography and dynamic light scattering (DLS) analysis demonstrated that the molecular size of native mmTRAIL was smaller than that of native hTRAIL. Cooling solutions of these proteins from room temperature to 0 A degrees C induced considerable precipitation of hTRAIL but not of mmTRAIL, indicating that mmTRAIL was more soluble than hTRAIL at low temperatures. Additionally, mmTRAIL was more resistant than hTRAIL to N-bromosuccinimide (NBS)-induced precipitation. Although mmTRAIL and hTRAIL showed comparable nanomolar affinities for human death receptors, the dissociation rate of the mmTRAIL-receptor complex was slower than that of the hTRAIL-receptor complex, suggesting that the mmTRAIL-receptor complex was more stable. Moreover, mmTRAIL induced caspase-dependent apoptosis in human tumor cells with an IC50 that was two to three times lower than that of hTRAIL. However, in vivo evaluation demonstrated that mmTRAIL or hTRAIL led to a similar level of tumor suppression in mice bearing COLO205 xenografts. Nevertheless, the advantage of its better solubility should promote the production and further use of mmTRAIL in cancer biotherapy.