Abstract
BackgroundTumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively eliminates tumor cells. However, the short biological half-life of this molecule limits its potential use in the clinic. Our aim was to construct a recombinant strain of nonpathogenic Lactococcus lactis bacteria as a vector for effective and prolonged human TRAIL production. Herein, we examined the expression and secretion conditions leading to the production of biologically active protein in vitro.ResultsThe human soluble TRAIL-cDNA (hsTRAIL-cDNA) with optimized codons was designed to fit the codon usage pattern (codon bias) of the L. lactis host. This cDNA construct was synthesized and cloned in lactococcal plasmid secretion vector pNZ8124 under the control of the nisin-induced PnisA promoter. The pNZ8124-hsTRAIL plasmid vector was transformed into the L. lactis NZ9000 host strain cells by electroporation. Secretion of the protein occurred at the neutral pH during induction, with optimized concentration of the inducer and presence of serine proteases inhibitor. Using Western blotting and amino acid sequencing method we found that TRAIL was secreted in two forms, as visualized by the presence of two distinct molecular size bands, both deprived of the usp45 protein, the bacterial signal peptide. By the use of MTS assay we were able to prove that hsTRAIL present in supernatant from L. lactis (hsTRAIL+) broth culture was cytotoxic to human HCT116 colon cancer cells but not to normal human fibroblasts. Flow cytometry analysis revealed TRAIL-induced apoptosis of cancer cells.ConclusionsWe designed recombinant L. lactis bacteria, which efficiently produce biologically active, anti-tumorigenic human TRAIL in vitro. Further studies in tumor-bearing NOD-SCID mice will reveal whether the TRAIL-secreting L. lactis bacteria can be used as a safe carrier of this protein, capable of inducing effective elimination of human colon cancer cells in vivo.
Highlights
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively eliminates tumor cells
In our study we propose L. lactis bacteria as the host for an efficient expression of a secretory bioactive form of human TRAIL
Lactococcus lactis bacteria require specific conditions for growth and efficient expression of recombinant hsTRAIL For the most effective expression of recombinant hsTRAIL in L. lactis bacteria, we designed a synthetic hsTRAIL-cDNA with optimized codons to fit the codon usage pattern of L. lactis as the host [18,19,20]
Summary
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively eliminates tumor cells. Tumor necrosis factor-related apoptosis-inducing ligand [3, 4] (TRAIL, other names: TNFSF10; CD253; Apo-2L; TNLG6A [5]), is a protein belonging to the TNF-superfamily [6] and has been shown in in vitro and in vivo models to induce apoptosis of various types of cancer cells while sparing normal ones [7]. TRAIL may act as a trans-membrane protein or can be cleaved from the cell surface by cathepsin E to form a soluble ligand. Both forms of TRAIL are biologically active and their interactions with specific death receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5) induce apoptosis of cancer cells upon activation of caspase-8 [4, 8]. Recent research on the new TRAIL formulations was focused on enhancing its bioactivity for cancer treatment and increasing its stability in humans [10]
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