Production and secretion of TNF related apoptosis inducing ligand (TRAIL/Apo2L) in the Escherichia coli periplasm using PhoA signal peptide

Abstract

The ability of TNF related apoptosis inducing ligand/Apo2 ligand (TRAIL/Apo2L) in order to selectively induce apoptosis in tumor cells but not normal cells makes it an attractive target for development of new cancer therapy. Although TRAIL/Apo2L has been produced in several hosts, E. coli is one of the best expression systems among them due to its safety, simplicity, low cultivation cost, and known genetic properties. However, cytoplasmic expression of TRAIL/Apo2L in E. coli may be concomitant with some problems such as protease-induced degradation, protein misfolding, diminution in the biological activity and complexity of downstream processing. Therefore, the aim of this study was the development of an expression system to produce and secrete recombinant TRAIL/Apo2L into the E. coli periplasmic space. DNA encoding Alkaline Phosphatase (PhoA) signal peptide was added to the TRAIL/Apo2L cDNA using overlapping extension PCR procedure. PhoA-TRAIL construct was subsequently cloned in pET-22b expression plasmid and correct cloning was confirmed by PCR and sequencing. TRAIL/Apo2L expression was induced in E. coli BL21 (DE3) and then its periplasmic fraction was isolated through osmotic shock. SDS-PAGE analysis showed that recombinant TRAIL/Apo2L was successfully secreted into E. coli periplasm. The periplasmic TRAIL/Apo2L was identified by western blotting analysis. Finally, the biological activity of the purified periplasmic TRAIL/Apo2L was assessed by MTT assay to evaluate its growth inhibitory effect against the HeLa cell line. In conclusion, the results demonstrate that our TRAIL/Apo2L expression system could be an interesting alternative to reduce problems arose from the cytoplasmic production of TRAIL/Apo2L.