Recombinant derivatives of the human high-mobility group protein HMGB2 mediate efficient nonviral gene delivery.
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 (HMG) protein HMGB2 and investigated their potential as novel protein-based transfection reagents. A truncated form of HMGB2 encompassing amino acids 1-186 of the molecule was expressed in Escherichia coli at high yield. This HMGB2186 protein purified from bacterial lysates was able to condense plasmid DNA in a concentration-dependent manner, and mediated gene delivery into different established tumor cell lines more efficiently than poly(l-lysine). By attaching, via gene fusion, additional functional domains such as the HIV-1 TAT protein transduction domain (TAT(PTD)-HMGB2186), the nuclear localization sequence of the simian virus 40 (SV40) large T-antigen (SV40(NLS)-HMGB2186), or the importin-beta-binding domain (IBB) of human importin-alpha (IBB-HMGB2186), chimeric fusion proteins were produced which displayed markedly improved transfection efficiency. Addition of chloroquine strongly enhanced gene transfer by all four HMGB2186 derivatives studied, indicating cellular uptake of protein-DNA complexes via endocytosis. The IBB-HMGB2186 molecule in the presence of the endosomolytic reagent was the most effective. Our results show that recombinant derivatives of human HMGB2 facilitate efficient nonviral gene delivery and may become useful reagents for applications in gene therapy.
Sloots
Arjen
A
Chemotherapeutisches Forschungsinstitut, Georg-Speyer-Haus, Frankfurt am Main, Germany.
Wels
Winfried S
WS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D018014:Gene Transfer Techniques; D024261:HMGB2 Protein / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D028901:alpha Karyopherins / Q000378:metabolism
D017931:DNA Primers; D024261:HMGB2 Protein; D011994:Recombinant Proteins; D028901:alpha Karyopherins