Exp Mol Med.  2008 Apr;40(2):151-160. 10.3858/emm.2008.40.2.151.

Membrane transducing activity of recombinant Hoxc8 protein and its possible application as a gene delivery vector

• Kim ES
• Kang M
• Park HW
• Kim MH

• KMID: 1052937
• DOI: http://doi.org/10.3858/emm.2008.40.2.151

Abstract

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 1 1 (DNA protein), and GST- Hoxc8(1-242) formed a complex at a mass ratio of 1 5. 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.

Keyword

Antennapedia homeodomain protein; gene transfer techniques; gene therapy; HOXC8 protein, human; transfection

MeSH Terms

Amino Acid Sequence
Animals
Base Sequence
Cell Line
DNA
DNA Primers
Electrophoresis, Agar Gel
*Genetic Vectors
Homeodomain Proteins/genetics/isolation & purification/*metabolism
Humans
Molecular Sequence Data
Recombinant Proteins/genetics/isolation & purification/metabolism
*Transduction, Genetic