Format

Send to

Choose Destination
J Microbiol Biotechnol. 2017 Dec 28;27(12):2156-2164. doi: 10.4014/jmb.1705.05070.

Soluble Prokaryotic Expression and Purification of Bioactive Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand.

Author information

1
Department of Physiology, Asan-Minnesota Institute for Innovating Transplantation, Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Asan Medical Center, Seoul 05505, Republic of Korea.
2
Department of Convergence Medicine, Asan Institute for Life Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 05505, Republic of Korea.
3
Department of Physiology, Cell Dysfunction Research Center, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea.

Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered as an antitumor agent owing to its ability to induce apoptosis of cancer cells without imparting toxicity toward most normal cells. TRAIL is produced in poor yield because of its insoluble expression in the cytoplasm of E. coli. In this study, we achieved soluble expression of TRAIL by fusing maltose-binding protein (MBP), b'a' domain of protein disulfide isomerase (PDIb'a'), or protein disulfide isomerase at the N-terminus of TRAIL. The TRAIL was purified using subsequent immobilized metal affinity chromatography and amylose-binding chromatography, with the tag removal using tobacco etch virus protease. Approximately 4.5 mg of pure TRAIL was produced from 125 ml flask culture with a purification yield of 71.6%. The endotoxin level of the final product was 0.4 EU/μg, as measured by the Limulus amebocyte lysate endotoxin assay. The purified TRAIL was validated and shown to cause apoptosis of HeLa cells with an EC₅₀ and Hill coefficient of 0.6 ± 0.03 nM and 2.41 ± 0.15, respectively. The high level of apoptosis in HeLa cells following administration of purified TRAIL indicates the significance and novelty of this method for producing high-grade and high-yield TRAIL.

KEYWORDS:

E. coli; HeLa cells; Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL); cytotoxic activity; expression; purification

PMID:
29032646
DOI:
10.4014/jmb.1705.05070
[Indexed for MEDLINE]
Free full text

Supplemental Content

Full text links

Icon for The Korean Society for Microbiology and Biotechnology.
Loading ...
Support Center