An archaeal histone-like protein as an efficient DNA carrier in gene transfer.
HPhA, a recombinant histone-like protein from Pyrococcus horikoshii OT3 strain, has compacting activity with DNA as previously reported. The extreme stability and DNA packaging activity of the HPhA make it a candidate as a DNA carrier. Here, the plasmid DNA-HPhA complexes were fully characterized by gel retardation assay and DNase resistance assay. It was further proved that HPhA has in vitro DNA transfection activity. HPhA-mediated transfection efficiency was dependent on the mass ratio of HPhA to DNA, the incubation time and the presence of calcium. A protocol for HPhA-mediated transfection in vitro was established to improve transfection efficiency. The optimal mass ratio of HPhA to DNA was 6:1, and the incubation time required for the DNA-HPhA complex to be in contact with the cell was 4 h. In addition, the presence of 2 mM CaCl2 in the cell culture medium was required for efficient transfection. Serum did not show inhibition of HPhA-mediated transfection. Most importantly, the cytotoxicity of HPhA is lower than that of commonly used cationic liposome-based gene delivery systems, and HPhA-mediated transfection in NIH 3T3, HEK 293, HL-7702, HepG2 and Cos 7 cell lines in vitro has a higher efficiency and reproducibility. These results demonstrate that the HPhA is a new, potentially widely applicable and highly efficient gene carrier.
Key Laboratory for Molecular Enzymology and Engineering, Ministry of Education, Jilin University, Changchun, 130023, PR China.
Liang
L
Weng
Dan
D
Liu
Yuanyuan
Y
Li
Shugui
S
Cao
Yan
Y
Feng
D000818:Animals; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D043902:Pyrococcus horikoshii / Q000378:metabolism; D044967:Serum / Q000378:metabolism; D014162:Transfection
D019843:Archaeal Proteins; D006657:Histones; D004247:DNA; D003850:Deoxyribonuclease I; D002118:Calcium