Synergistic effect of granzyme B-azurin fusion protein on breast cancer cells.

As one of the most prevalent malignancies, breast cancer still remains a significant risk for public health. Common therapeutic strategies include invasive surgery, chemotherapy and anti-herceptin antibodies. Adverse effects, drug resistance and low efficacy of current therapies necessitates the emergence of more effective platforms. Naturally released by the immune system, granzyme B activates multiple pro-apoptotic pathways by cleaving critical substrates. Bacterial cupredoxin, azurin, selectively targets cancer cells via a p53-dependent pathway. Fused by a linker, GrB-Azurin fusion protein was overexpressed in HEK293T cells, and purified by metal chromatography. SDS-PAGE, Western blotting and ELISA were performed to confirm successful expression, purification and analyze binding properties of the fusion protein. After treatment of various breast cancer cell lines with increasing concentrations of GrB-Azurin, quantitative real-time RT-PCR was used to measure relative expression of p21, Fas and DR5 pro-apoptotic genes. The results of DNA fragmentation and WST-1 cell viability assays indicated significant apoptosis induction in MDA-MB-231, MCF7 and SK-BR-3 cells, while insignificant cytotoxicity was detected on MCF 10A normal breast cells. Herein, we report the development of a novel biotherapeutic against breast cancer. Selective effectiveness of GrB-Azurin fusion protein on different breast cancer cells highlighted the potential of the designed construct as a candidate anti-cancer biodrug.

Rational design-based engineering of a thermostable phytase by site-directed mutagenesis.

Phytases are enzymes that hydrolysis phytic acid and makes mineral phosphorus available to animals. Phytases face relatively extreme heating during food processing, thus thermostability plays an important role in industrial applicability of this enzyme. Herein, we report the design of a thermostable phytase with favorable biochemical properties and high enzymatic activity using molecular dynamics and rational design-based molecular engineering. Based on the crystal structure of E. coli phytase, bioinformatics analysis and docking binding energy measurement, S392F mutant was introduced by site-directed mutagenesis in order to improve thermostability of phytase through strengthen the bounding interactions. Wilde type and Mutated constructs were expressed in E. coli BL 21. The WT and manipulated phytase were purified; their biochemical and kinetic was investigated. Results revealed that recombinant WT and mutant phytase have optimum temperature of 50 °C with no significance change but optimum pH of WT and mutant was respectively 5 and 6 with a pH shift. Furthermore, S392F phytase catalytic efficiency values showed significant improve of 25.6%, compared with the WT. Analysis of the retained enzymatic activity at high temperatures, indicated that despite of phytase stability reduction at high temperatures but mutant phytase showed more stable behavior in compare with WT phytase, So that at 70 °C showed twice thermo stability and at 80 °C and 90 °C display respectively 74% and 78.4% improvement of thermostability compared to the wild-type. In conclusion, our results implied that the designed phytase could be a potential candidate for phytase manipulation research and industrial applications with improved thermostability.