Expression and characterization of a recombinant high mobility group box 1 AB peptide with a 6-histidine tag for delivery of nucleic acids

• Published in: Enzyme and microbial technology 2008-11, Vol.43 (6), p.410-416
• Main Authors: Kim, Kyunghwa, Han, Jee Seung, Park, Jeong Hyun, Ko, Kyung Soo, Lee, Minhyung
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Biotechnology; Cytotoxicity; Fundamental and applied biological sciences. Psychology; Gene delivery; High mobility group box 1; Recombinant DNA; Transfection
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/j.enzmictec.2008.07.002

In this research, a recombinant high mobility group box 1 AB peptide with a 6-histidine tag (rHMGB1AB-His6) was produced and characterized as a carrier of nucleic acids. The human HMGB1AB cDNA was cloned by RT-PCR. The rHMGB1AB-His6 expression vector, pET21a-rHMGB1AB-His6, was constructed with the cDNA. In pET21a-rHMGB1AB-His6, the acidic box of the C-terminus of wild type HMGB1 was eliminated, since it had negative charges and interfered with the DNA and peptide interaction. pET21a-rHMGB1AB-His6 was transformed into the BL21 strain. rHMGB1AB-His6 was produced by IPTG induction and purified using a nickel column. A gel retardation assay showed that plasmid DNA (pDNA) was completely retarded at a 1:1 weight ratio. The complex size was approximately 150 nm at a 1:10 weight ratio (pDNA/rHMGB1AB-His6) and had a tendency to increase with increasing amount of rHMGB1AB-His6. The cytotoxicity of rHMGB1AB-His6 was compared with poly- l-lysine (PLL, 20 kDa). As a result, rHMGB1AB-His6 did not show any cytotoxicity to 293 cells, while PLL had a greater cytotoxicity. rHMGB1AB-His6 had the highest transfection efficiency at a 1:40 weight ratio (pDNA/rHMGB1AB-His6). In addition, rHMGB1AB-His6 showed comparable transfection efficiency to PLL. With low cytotoxicity and moderate transfection efficiency, rHMGB1AB-His6 may be useful for delivery of nucleic acids.