Membrane transducing activity of recombinant Hoxc8 protein and its possible application as a gene delivery vector.
In order to examine whether the Hoxc8 protein can deliver nucleic acid into mammalian cells, we designed several Hoxc8-derived recombinant proteins to be synthesized as glutathione S-transferase (GST) fused forms in E. coli (GST-Hoxc8(1-242), containing a full length of Hoxc8; GST-Hoxc8(152-242), possessing a deletion of the acidic N-terminus of Hoxc8; GST-Hoxc8(149-208), which contained the homeodomain only). After labeling these proteins with Oregon 488, we examined their membrane transduction ability under the fluorescence microscope and verified that all three proteins showed similar transduction efficiency. The ability of the proteins to form in vitro protein-DNA complexes was analyzed on agarose gel; both GST-Hoxc8(1-242) and GST-Hoxc8(149-208) formed complexes. In contrast, the GST-Hoxc8(152-242) protein did not form a complex. The GST-Hoxc8(149-208) protein formed a complex with DNA at a mass ratio of 1ú1 (DNAúprotein), and GST-Hoxc8(1-242) formed a complex at a mass ratio of 1ú5. When the DNA (pDsRed1-C1) and protein complexes were added to culture media containing mammalian cells, the cells uptook the complexes, which was indicated by red fluorescence expression under the fluorescent microscope. These results indicate that recombinant Hoxc8 derivatives that harbor a homeodomain are able to traverse the mammalian cellular membrane. DNA that is bound to the recombinant derivatives can be carried across the membrane as well. This process could be applied in the development of a useful delivery vector for gene therapy in the future.
Kim
Eun Shin
ES
Department of Anatomy, Embryology Laboratory, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Korea. mhkim1@yuhs.ac
Kang
Myengmo
M
Park
Hyoung Woo
HW
Kim
Myoung Hee
MH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D005822:Genetic Vectors; D018398:Homeodomain Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014161:Transduction, Genetic
D017931:DNA Primers; C489815:HOXC8 protein, human; D018398:Homeodomain Proteins; D011994:Recombinant Proteins; D004247:DNA