학술논문
Secretion of tumoricidal human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by recombinant Lactococcus lactis: optimization of in vitro synthesis conditions
Document Type
Report
Author
Source
Microbial Cell Factories. November 16, 2018, Vol. 17 Issue 1
Subject
Language
English
ISSN
1475-2859
Abstract
Author(s): Katarzyna CiaÄma[sup.1] , Jerzy WiÄckiewicz[sup.1] , Sylwia KÄdracka-Krok[sup.2] , Magdalena Kurtyka[sup.3] , MaÅgorzata Stec[sup.1] , Maciej Siedlar[sup.1] and Jarek Baran[sup.1] Background Colorectal cancer is one of the most common [...]
Background Tumor 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. Results The 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. Conclusions We 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. Keywords: TRAIL, Lactococcus lactis, Nisin Controlled Gene Expression System, Colorectal cancer
Background Tumor 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. Results The 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. Conclusions We 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. Keywords: TRAIL, Lactococcus lactis, Nisin Controlled Gene Expression System, Colorectal cancer