Periplasmic Expression of TNF Related Apoptosis Inducing Ligand (TRAIL) in E.coli

Area of Science:

• Biotechnology
• Molecular Biology
• Cancer Research

Background:

• Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) shows promise for cancer therapy due to its selective induction of apoptosis in tumor cells.
• Recombinant protein expression in E. coli cytoplasm often results in misfolding, inclusion bodies, and reduced biological activity, complicating downstream processing.

Purpose of the Study:

• To develop an improved expression system for producing recombinant TRAIL.
• To achieve secretion of functional TRAIL into the E. coli periplasm, avoiding cytoplasmic expression issues.

Main Methods:

• A signal sequence (OmpA) was fused to TRAIL cDNA using Overlapping Extension PCR.
• The OmpA-TRAIL fragment was cloned into the pET-22b plasmid and expressed in E. coli BL21 (DE3).
• Periplasmic proteins were isolated via osmotic shock, and expression was analyzed by SDS-PAGE and Western blot; cytotoxic activity was assessed using MTT assay.

Main Results:

• The OmpA signal sequence facilitated the secretion of recombinant TRAIL into the E. coli periplasm.
• Approximately 37% of the expressed recombinant TRAIL was successfully translocated to the periplasm.
• The periplasmic TRAIL retained its identity and demonstrated cytotoxic activity against HeLa cells.

Conclusions:

• The developed expression system enables the efficient production of recombinant TRAIL in the E. coli periplasm.
• This periplasmic secretion strategy effectively overcomes challenges associated with cytoplasmic expression, such as misfolding and inclusion body formation.
• The system holds potential for the scalable production of biologically active TRAIL for therapeutic applications in cancer treatment.