融合蛋白RGD-TRAIL在毕赤酵母中的表达及活性

Expression and antitumor activity of fusion protein RGD-TRAIL in Pichia pastoris

本文分别利用大肠杆菌和毕赤酵母表达系统构建表达RGD-TRAIL蛋白,比较两种表达系统对该蛋白活性的影响,并对毕赤酵母表达的RGD-TRAIL进行活性分析。利用基因工程方法分别构建重组质粒p ET30-rgdtrail和p HBM-rgd-trail,于大肠杆菌和毕赤酵母中诱导表达。采用MTT法、ELISA法、划痕及Transwell小室和Hoechst 33342染色检测RGD-TRAIL对肿瘤细胞增殖抑制、亲和活性、迁移和凋亡的影响。Western blotting检测相关凋亡蛋白的表达。重组蛋白RGD-TRAIL于大肠杆菌中以包涵体形式表达,在毕赤酵母中分泌表达。MTT结果显示,毕赤酵母表达的RGD-TRAIL抑制肿瘤细胞增殖活性明显强于大肠杆菌表达的RGD-TRAIL。ELISA实验表明,酵母表达的RGD-TRAIL可与表达αv和DR4、DR5的肿瘤细胞发生特异性结合。划痕和Transwell实验证明,RGD-TRAIL能抑制肿瘤细胞A549和HT1080迁移。经过RGD-TRAIL作用后,荧光显微镜下可观察到肿瘤细胞明显发生凋亡,Western blotting检测PARP及caspase-3均出现剪接体。研究结果提示,毕赤酵母表达系统更适合于RGD-TRAIL蛋白的表达,且能够很好地发挥靶向作用及杀伤活性。

To compare the activity of RGD-TRAIL in different expression systems, RGD-TRAIL in both Escherichia coli （E.coli） and Pichia pastoris was constructed and expressed. In vitro activity of RGD-TRAIL from Pichia pastoris expression system was also analyzed. Genetic engineering techniques were used to construct recombinant plasmid pET30-rgd-trail and pHBM-rgd-trail. The recombinant protein RGD-TRAIL was purified with Ni ion affinity chromatography after induction. MTT assay, ELISA, scratch wound healing, transwell migration assay and Hoechst 33342 staining were performed to detect the effects of RGD-TRAIL on proliferation, binding activity, migration and apoptosis. The expression of apoptosis-associated proteins was detected by Western blotting. Recombinant protein RGD-TRAIL was successfully expressed in a form of inclusion body in E.coli, while expressed secretorily in Pichia pastoris. It possessed more potent cytotoxicity than RGD-TRAIL in E.coli by MTT assay. The RGD-TRAIL expressed by Pichia pastoris showed powerful binding affinity with cancer cells expressing av, DR4, DR5 and highly potent cytotoxicity through inducing apoptosis of cancer cells. Nuclear fragmentation was examined by Hoechst 33342 staining. Cleaved PARP and caspase-3 were also detected after incubation with RGD-TRAIL. Additionally, RGD-TRAIL inhibited migration significantly in A549 and HT1080 ceils. The results demonstrate that Pichia pastoris expression system is more suitable for the recombinant protein RGD-TRAIL. Its binding affinity and antitumor activity might make RGD-TRAIL a promising candidate for cancer therapy.