Cloning and recombinant expression of human soluble TRAIL in Pichia pastoris.
- Author:
Yingying LI
1
;
Lin WAN
;
Hao YANG
;
Shan LIU
;
Huawei CAI
;
Xiaofeng LU
Author Information
1. Life Sciences College, Sichuan University, Chengdu 610041, China.
- Publication Type:Journal Article
- MeSH:
Antineoplastic Agents;
pharmacology;
Cell Line, Tumor;
Cloning, Molecular;
Cytotoxicity Tests, Immunologic;
methods;
Genetic Vectors;
genetics;
Humans;
Pichia;
genetics;
metabolism;
Recombinant Proteins;
biosynthesis;
genetics;
pharmacology;
TNF-Related Apoptosis-Inducing Ligand;
biosynthesis;
genetics
- From:
Journal of Biomedical Engineering
2010;27(6):1307-1326
- CountryChina
- Language:Chinese
-
Abstract:
TNF-related apoptosis-inducing ligand (TRAIL) is a member of factor TNF family, which could be potentially developed as novel antitumor agent due to its selective and efficient induction of apoptosis in tumor cells. Gene recombinant expression is an important tool for production of pharmaceutical protein. In this paper, the gene encoding human soluble TRAIL (114-281aa fragment) was cloned by PCR and then inserted into the Pichia Pastoris expression vector pPIC9K. The transformants were double-screened on plates containing neomycin G418 and many clones with high levels of G418-resistance were selected for further studies on protein expression. The recombinant human soluble TRAIL was secreted into the BMMY media under the condition of 3% methanol. And the recombinant protein was purified to homogeneity (-80% purity) by using Ni-agarose affinity chromatography. The yield of this protein is about 1-2 mg per liter culture. Cell viability assays demonstrated that human soluble TRAIL was cytotoxic in both leukemia cells Jurkat and lung cancer cells A549. After treatment with 0.05 microg/ml TRAIL, the survival rate of Jurkat cells was about 10%. The expressed TRAIL showed dose-dependent cytotoxicity in A549 cells within the range of 0.1-1 microg/ml. When the protein concentration reached 1 microg/ml, the survival rates of A549 cells were about 30%. However, the recombinant human soluble TRAIL did not show obvious cytotoxicity in human skin fibroblast cells (HSF) at concentrations tested. There results demonstrate that human soluble TRAIL is selectively cytotoxic in tumor cells. The expression system constructed in this experiment might contribute to further production of soluble TRAIL and TRAIL-based novel fusion proteins in large quantities.
