Construction, Expression, and Purification of p28 as a Cell-Penetrating Peptide ‎with Anticancer Effects on Burkitt’s Lymphoma Cell Line

Abstract

Background: The p28 is a small-sized cell-penetrating peptide derived from bacterial protein azurin and can function as a cancer-specific anti-proliferative agent. It can penetrate cancer tissues easily without involving the immune system, and increase the intracellular concentration of p53. Objectives: In this study, we have expressed and purified recombinant p28, then evaluated its anti-proliferative and pro-apoptotic effects on Raji cancer cell line. Methods: The p28 gene was amplified and cloned into pTZ57R cloning vector and was sequenced subsequently. Afterward, it was transformed into E. coli BL21 bacterial host by using pET-28a expression vector. Peptide purification was carried out using Ni-NTA ‎chromatography system. Bradford, SDS-PAGE, and western blotting assays were applied to assess the concentration and expression level of the recombinant peptide. The proficiency ‎of p28 in inhibition of tumor growth and induction of apoptosis in cancerous cells was investigated by evaluating the Raji and HEK-293 cells treated with different concentrations of p28. Results: The overexpression of the p28 peptide in the bacterial host was confirmed by SDS-PAGE and western blotting. Moreover, Bradford assay revealed desirable concentrations of the recombinant p28 before and after dialysis. The MTT and PE-Annexin V apoptosis assays indicated the specific function of p28 in impeding the proliferation of cancerous cells and triggering the apoptosis. Conclusions: The p28 induces apoptosis in cancerous cells but not in normal control cells. ‎In summary, p28 is a non-immunogenic small peptide that can penetrate cancerous cells ‎preferentially, impede the cell proliferation, and induce the apoptosis. Overall, these ‎findings suggest p28 as‎ a promising anticancer drug.