Development and Bioactivity Assessment of a Recombinant Pseudomonas aeruginosa Azurin-BR2 Peptide Fusion Protein: A Novel Approach to Cancer Immunotherapy

Abstract

Background: Conventional chemotherapeutic drugs have limitations, including non-specific toxicity and drug resistance. Targeted cancer therapy using recombinant fusion proteins may effectively overcome these challenges. Azurin is a bacteriocin with pro-apoptotic activity, while buforin IIb derivative (BR2) is an antimicrobial peptide exhibiting cell-penetrating ability. This study aims to construct a fusion protein combining Azurin and BR2 to explore its potential against breast cancer cell line (MCF-7). Methods: We designed, expressed, purified, and evaluated a novel recombinant fusion protein named Azurin-BR2, consisting of Azurin and BR2 domains, for targeted cytotoxicity against breast cancer cells. The fusion gene construct was cloned into a pET30a vector and transformed into E. coli BL21DE3 for expression. The fusion protein was isolated from inclusion bodies, solubilized, and refolded. The purity and identity of the 19 kDa fusion protein were confirmed by Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western blot analysis. To evaluate its biological activity, the fusion protein was tested using the 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide (MTT) assay on both MCF-7 breast cancer cells and MCF-10F normal cells. Results: The fusion protein showed significant cytotoxic effects against MCF-7 cells, outperforming Azurin alone. The cytotoxic impact on normal MCF-10F cells was significantly lower. The half-maximal inhibitory concentration (IC₅₀) value for Azurin-BR2 was 52 μg/mL compared to 102 μg/mL for Azurin alone against MCF-7 cells. Conclusions: The fusion protein Azurin-BR2 demonstrated enhanced anticancer therapeutic efficacy by combining the pro-apoptotic activity of Azurin with the cell-penetrating properties of BR2. This fusion protein holds promise for targeted cancer therapy in clinical applications and warrants further in vivo studies.