Recombinant high mobility group box-1 peptide for delivery of nucleic acids

Abstract

High mobility group box-1 (HMGB-1) is an abundant nuclear protein. HMGB1 is composed of high mobility group (HMG) box A, box B, and acidic C-terminal region. Boxs A and B are DNA binding domains and have high conents of basic amino acids. In this study, a recombinant HMGB-1 box A (HMGB-1A) peptide was produced as a carrier of nucleic acids. The HMGB-1A cDNA was amplified by RT-PCR using total RNA from 293 cells as a template. The HMGB-1A cDNA was cloned into the pET21a expression vector, resulting in construction of pET21a-HMGB-1A. pET21a-HMGB-1A was transfromed into the BL21 strain and HMGB-1A was over-expressed by IPTG induction. HMGB-1A was purified using nickel affinity and ion exchange chromatography. The purified HMGB-1A was analyzed in SDS-PAGE. Gel retardation assay showed that HMGB-1A formed complexes with plasmid DNA or siRNA effectively. In transfection assay, HMGB-1A delivered luciferase plasmid DNA into 293 cells. The transfection efficiency of HMGB-1A was comparable to that of poly-L-lysine (PLL). In addition, HMGB-1A delivered anti-luciferase siRNA in CT-26 cells and reduced the internal luciferase expression. In MTT assay, HMGB-1A protein did not have any toxicity to cells, while PLL had much higher toxicity. Therefore, HMGB-1A may be useful for development of a non-toxic gene delivery carrier.