Recombinant Production and Intein-Mediated Purification of an Antimicrobial Peptide, BR2

Abstract

Nowadays, targeted therapy of cancer is under intensive focus of many investigations due to severe side effects imposed by various cancer chemotherapeutics. BR2 is a modified antimicrobial cell penetrating peptide with confirmed capability of delivering various cargos specifically to cancerous cells. However, because of its small size, its recombinant production by conventional methods is difficult, and its chemical synthesis imposes high cost. Hence, the aim of the present study was to evaluate if recombinant production and intein-mediated purification of this peptide is possible and finally evaluate its safety as a suitable targeted drug delivery vector on cancer and normal cell lines. In this regard, the coding sequence of BR2 was cloned in pTBX1 to be expressed in-frame with GyrA intein and then subjected to inducible protein expression using IPTG. Afterwards, the expressed protein was transferred to chitin-loaded columns and the peptide was purified according to manufacrure's instruction. SDS-PAGE and Western blot analysis confirmed the expression of BR2-GyrA fusion protein by showing a band of approximately 31 kDa. Moreover, SDS-PAGE of the purified peptide showed a band of approximately 3 kDa, confirming the successful purification of BR2. Finally, in order to evaluate the safety of the produced peptide, its effects was evaluated on MCF-7 and HEK-293 cell lines by MTT assay, and compared to the effects of chemically synthesized BR2. Statistical analysis of the MTT assay results showed that the recombinantly produced peptide had no significant toxic effects on MCF-7 and HEK 293 cells, comparing to negative control, and this was similar to the effects of the synthetic BR2. Hence, the recombinant BR2 can be used for production of novel vehicles for targeted delivery of cytotoxic cargos in the future.