Expression, purification and characterization of TAT-high mobility group box-1A peptide as a carrier of nucleic acids.
High mobility group box 1 (HMGB1) is an abundant nuclear protein that binds to double-stranded DNA. HMGB1 is composed of high mobility (HMG) box A, box B, and C-terminal acidic regions. In this study, a recombinant TAT linked HMGB1 box A (rTAT-HMGB1A) peptide was expressed, purified, and characterized as a carrier of nucleic acids. The HMGB1A cDNA was amplified by PCR, and cloned into the pET21a expression vector with the TAT domain located at the N-terminus. The rTAT-HMGB1A peptide was overexpressed and purified using Nickel affinity chromatography. A recombinant HMGB1A (rHMGB1A) peptide without the TAT domain was also overexpressed and purified as a control. In gel retardation assays, both the rHMGB1A and rTAT-HMGB1A peptides formed complexes with DNA equally well. However, transfection assays showed that the rTAT-HMGB1A peptide had a higher gene transfer efficiency than rHMGB1A. Finally, rTAT-HMGB1A had no cytotoxicity to HEK 293 cells suggesting that rTAT-HMGB1A may be useful as a non-toxic gene delivery carrier.
Kim
Kyunghwa
K
Department of Bioengineering, College of Engineering, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791, Republic of Korea.
Han
Jee Seung
JS
Kim
Hyun Ah
HA
Lee
Minhyung
M
D016923:Cell Death; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D015696:Gene Products, tat / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D024243:HMGB1 Protein / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D009696:Nucleic Acids / Q000378:metabolism*; D010316:Particle Size; D010455:Peptides / Q000302:isolation & purification* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D015696:Gene Products, tat; D024243:HMGB1 Protein; D009696:Nucleic Acids; D010455:Peptides; D011993:Recombinant Fusion Proteins