Recombinant Escherichia coli strain C600/pBV-TRAIL (encoding for 114-281 amino acids of TRAIL's soluble fragment) produced a recombinant human tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL). Using a combined strategy of exponential feeding and pH-stat feeding, high concentrations of biomass (65 g dry wt l(-1)) and active soluble TRAIL (1.4 g l(-1)) were obtained within 30 h. The accumulation of acetate, which usually occurs during the process of high-density culture of Escherichia coli and especially in the induction stage of protein synthesis, was avoided.

Interleukin-24 (IL-24) is a unique IL-10 family cytokine that could selectively induce apoptosis in cancer cells without harming normal cells. Previous research demonstrated that intracellular IL-24 protein induces an endoplasmic reticulum (ER) stress response only in cancer cells, culminating in apoptosis. In this study, we developed a novel recombinant fusion protein to penetrate into cancer cells and locate on ER. It is composed of three distinct functional domains, IL-24, and the targeting domain of transactivator of transcription (TAT) and an ER retention four-peptide sequence KDEL (Lys-Asp-Glu-Leu) that link at its NH2 and COOH terminal, respectively. The in vitro results indicated that TAT-IL-24-KDEL inhibited growth in bladder cancer cells, as well as in non-small cell lung cancer cell line and breast cancer cell line, but the normal human lung fibroblast cell line was not affected, indicating the cancer specificity of TAT-IL-24-KDEL. Western blot analysis showed that apoptosis activation was induced by TAT-IL-24-KDEL through the ER stress-mediated cell death pathway. Treatment with TAT-IL-24-KDEL significantly inhibited the growth of human H460 xenografts in nude mice, and the tumor growth inhibition was correlated with increased hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. These findings suggest that the artificially designed recombinant fusion protein TAT-IL-24-KDEL may be highly effective in cancer therapy and worthy of further evaluation and development.

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Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) is a new member of the TNF superfamily. In this work, the key role of Zn(2+) in high-level expression of soluble TRAIL was confirmed. The yield of soluble TRAIL reached 1.6 g l(-1) using a novel, two-stage Zn(2+) feeding strategy, and the accumulation of TRAIL inclusion bodies decreased. Furthermore, the purified TRAIL showed stronger cytotoxicity activity against human pancreatic 1990 tumor cells as the molar ratio of Zn(2+) to TRAIL monomer was 2 in purified TRAIL solution.

Immunotoxins are a new class of antibody-targeted therapy in clinical development. Traditional immunotoxins that are constructed from the toxins of plants or bacteria need to be internalized to the cytoplasm and thus have limited antitumor efficacy. In the present study, we combined a recently reported sea anemone cytolysin Gigantoxin-4 with an anti-HER2/neu single-chain variable fragment 4D5 scFv to construct a novel immunotoxin. We fused a SUMO tag to the N-terminus of Gigantoxin-4-4D5 scFv and it was successfully expressed in Escherichia coli strain BL21 (DE3) in a soluble form. After purification, the purity of Gigantoxin-4-4D5 scFv reached 96 % and the yield was 14.3 mg/L. Our results demonstrated that Gigantoxin-4-4D5 scFv exerted a highly cytotoxic effect on the HER2/neu-positive ovarian carcinoma SK-OV-3 cell line. And the hemolytic activity was weaker, making it safe for normal cells. The results of immunofluorescence analysis showed that this novel immunotoxin could specifically bind to SK-OV-3 cells with no recognition of human embryonic kidney 293 cells. Scanning electron microscope observations and extracellular lactate dehydrogenase activity indicated that it could induce necrosis in SK-OV-3 cells by disrupting the cell membrane. Moreover, it could also mediate apoptosis of SK-OV-3 cells.