Farokhinejad Fahimeh, Behbahani Abbas Behzad, Rafiei Dehbidi Gholam Reza, Takhshid Mohammad Ali
Department of Medical Biotechnology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran; Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
Protein Expr Purif. 2017 Oct;138:25-33. doi: 10.1016/j.pep.2017.07.004. Epub 2017 Jul 13.
N-myc downstream regulated gene2 (NDRG2) belongs to tumor suppressor protein family of NDRG. Anti-proliferative and anti-metastasis of NDRG2 overexpression has been demonstrated in a number of tumors. The aim of this study was to fuse the gene of Trans Activator of Transcription (TAT) protein transduction domain with NDRG2 gene and express and purify TAT-NDRG2 fusion protein in order to investigate the effects of TAT-NDRG2 protein on proliferation and apoptosis of LNCaP prostate carcinoma cell line. pET28a-TAT-NDRG2 and pET28a-NDRG2 plasmids were constructed and transformed into E. coli-BL21(DE3). TAT-NDRG2 and NDRG2 proteins were expressed in the bacteria, purified using affinity chromatography and verified using western blotting. The effects of TAT-NDRG2 and NDRG2 protein treatment on LNCaP cells proliferation and apoptosis were evaluated using MTT assay and AnnexinV, 7-AAD flow cytometry assay, respectively. Western blot analysis confirmed the expression and purification of TAT-NDRG2 and NDRG2 proteins. Treatment of LNCaP cells with TAT-NDRG2 protein increased cell death and induced apoptosis significantly (P < 0.05) compared to control and NDRG2 protein-treated cells. These results suggest that TAT-NDRG2 protein can be considered as a therapeutic modality for the treatment of tumors.
N-myc下游调控基因2(NDRG2)属于NDRG肿瘤抑制蛋白家族。NDRG2过表达的抗增殖和抗转移作用已在多种肿瘤中得到证实。本研究的目的是将转录反式激活因子(TAT)蛋白转导结构域基因与NDRG2基因融合,表达并纯化TAT-NDRG2融合蛋白,以研究TAT-NDRG2蛋白对LNCaP前列腺癌细胞系增殖和凋亡的影响。构建了pET28a-TAT-NDRG2和pET28a-NDRG2质粒,并将其转化到大肠杆菌BL21(DE3)中。TAT-NDRG2和NDRG2蛋白在细菌中表达,通过亲和层析纯化,并用蛋白质免疫印迹法进行验证。分别使用MTT法和AnnexinV、7-AAD流式细胞术检测法评估TAT-NDRG2和NDRG2蛋白处理对LNCaP细胞增殖和凋亡的影响。蛋白质免疫印迹分析证实了TAT-NDRG2和NDRG2蛋白的表达和纯化。与对照和NDRG2蛋白处理的细胞相比,用TAT-NDRG2蛋白处理LNCaP细胞可显著增加细胞死亡并诱导凋亡(P<0.05)。这些结果表明,TAT-NDRG2蛋白可被视为一种肿瘤治疗方式。