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

Abstract

Certain natural peptides and proteins of mammalian origin are able to bind and condense plasmid DNA, a prerequisite for the formation of transfection-competent complexes that facilitate nonviral gene delivery. Here we have generated recombinant derivatives of the human high-mobility group B-1 (HMGB-1). In the nucleus, HMGB-1 is an architectural chromatin-binding factor that bends DNA and promotes protein assembly on specific DNA targets. HMGB is structurally composed of three different domains: two homologous DNA-binding sequences entitled box domain (A and B) and a long acidic C-terminal domain. They bind DNA without sequence specificity, but have a high affinity for bent linear DNA. In this research, we produced a recombinant HMGB-1 without the acidic domain as a gene delivery carrier, since the acidic domain has negative charges and may decrease the interaction between HMGB-1 and DNA. The HMGB-1 cDNA was amplified by RT-PCR using total RNA from 293 cells as a template. The cDNA encoding human HMGB-1 without the acidic domain was subcloned into the expression vector pET-21a. The construction plasmid was introduced into the E.coli BL21, and the recombinant HMGB-1 was overproduced after IPTG induction. The expressed recombinant HMGB-1 was purified from the harvested cells through ultra-sonication and nickel-chelating column chromatography. The purified HMGB-1 exhibited a molecular mass of 22 kD in SDS-PAGE analysis. Gel retardation assay showed that HMGB-1/DNA complex was completely retarded at a 1:1 weight ratio. In vitro transfection assay showed that HMGB-1 showed the highest transfection efficiency at a 30/1 weight ratio (HMGB-1/DNA). In addition, the cytotoxicity of HMGB-1 was lower than poly-L-lysine. Our results show that a recombinant derivate of human HMGB facilitates nonviral gene delivery and may be a useful reagent for application in gene therapy.