Expression and antitumor activity of fusion protein RGD-TRAIL in Pichia pastoris.
- Author:
Xiao-fei WANG
;
Shu-zhen CHEN
- Publication Type:Journal Article
- MeSH:
Antineoplastic Agents;
pharmacology;
Apoptosis;
Blotting, Western;
Cell Line, Tumor;
Chromatography, Affinity;
Enzyme-Linked Immunosorbent Assay;
Escherichia coli;
Humans;
Oligopeptides;
biosynthesis;
pharmacology;
Pichia;
metabolism;
Plasmids;
Recombinant Fusion Proteins;
biosynthesis;
pharmacology;
TNF-Related Apoptosis-Inducing Ligand;
biosynthesis;
pharmacology
- From:
Acta Pharmaceutica Sinica
2015;50(5):552-559
- CountryChina
- Language:Chinese
-
Abstract:
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 α(v), 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 cells. 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.
