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.

No abstract

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.

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.

Aim:  To improve phenylalanine ammonia lyase (E.C.4·3·1·5‐PAL) activity in recombinant Escherichia coli. Some methods for enrichment of PAL activity in recombinant E. coli JM109 were described. In an effort to create a rich enzyme source these methods would lead to improvements in the production of l‐phenylalanine. Methods and Results:  The possibilities of enriching PAL activity in recombinant E. coli was investigated by using individual and combinations of amino acids, organic solvents and surfactants. PAL activity was induced by adding combination of l‐phenylalanine and l‐tyrosine, activities as high as 64·3 U g−1of cells were obtained and enzyme activity was enriched by over 3·5‐fold in comparison with the control. Permeabilization with cetyl trimethyl ammonium bromide or the acetone significantly enriched cellular PAL activity, which improved over 8·2‐ and 9·0‐fold compared with the control, as high as 148·5 and 164·5 U g−1of cells respectively. Conclusion:  These efforts may provide some effective methods for enhancing l‐phenylalanine ammonia lyase activity. Significance and Impact of the Study:  These approaches for manipulating recombinant E. coli in an effort to create a rich enzyme source would serve as a biotechnologically important protocol for production of l‐phenylalanine.

Prostate apoptosis response-4 (Par-4), an anticancer protein that interacts with cell surface receptor GRP78, can selectively suppress proliferation and induce apoptosis of cancer cells. The core domain of Par-4 (aa 137-195), designated as SAC, is sufficient to inhibit tumor growth and metastasis without harming normal tissues and organs. Nevertheless, the anticancer effects of SAC have not been determined in ovarian cancer cells. Here, we developed a novel method for producing native SAC in Escherichia coli using a small ubiquitin-related modifier (SUMO) fusion system. This fusion system not only greatly improved the solubility of target protein but also enhanced the expression level of SUMO-SAC. After purified by Ni-NTA affinity chromatography, SUMO tag was cleaved from SUMO-SAC fusion protein using SUMO protease to obtain recombinant SAC. Furthermore, we simplified the purification process by combining the SUMO-SAC purification and SUMO tag cleavage into one step. Finally, the purity of recombinant SAC reached as high as 95% and the yield was 25 mg/L. Our results demonstrated that recombinant SAC strongly inhibited proliferation and induced apoptosis in ovarian cancer cells SKOV-3. Immunofluorescence analysis and competitive binding reaction showed that recombinant SAC could specifically induce apoptosis of SKOV-3 cells through combination with cell surface receptor, GRP78. Therefore, we have developed an effective strategy for expressing bioactive SAC in prokaryotic cells, which supports the application of SAC in ovarian cancer therapy.

Interleukin-24 (IL-24) is a cytokine belonging to the IL-10 gene family. This cytokine selectively induces apoptosis in cancer cells, without harming normal cells, through a mechanism involving endoplasmic reticulum (ER) stress response. TAT-IL-24-KDEL is a fusion protein that efficiently enters the tumor cells and locates in the ER. Here we report that TAT-IL-24-KDEL induced apoptosis in human cancer cells, mediated by the ER stress cell death pathway. This process was accompanied by the inhibition of the transcription of an antiapoptotic protein, survivin. The forced expression of survivin partially protected cancer cells from the induction of apoptosis by TAT-IL-24-KDEL, increased their clonogenic survival, and attenuated TAT-IL-24-KDEL-induced activation of caspase-3/7. RNA interference of survivin markedly sensitized the transformed cells to TAT-IL-24-KDEL. Survivin was expressed at higher levels among isolated clones that resistant to TAT-IL-24-KDEL. These observations show the important role of survivin in attenuating cancer-specific apoptosis induced by TAT-IL-24-KDEL. The pharmacological inhibition of survivin expression by a selective small-molecule survivin suppressant YM155 synergistically sensitized cancer cells to TAT-IL-24-KDEL-induced apoptosis in vitro and in vivo. The combined regimen caused significantly higher activation of ER stress and dysfunction of mitochondria than either treatment alone. As survivin is overexpressed in a majority of cancers, the combined TAT-IL-24-KDEL and YM155 treatment provides a promising alternative to the existing therapies.