Recombinant fusion proteins TAT-Mu, Mu and Mu-Mu mediate efficient non-viral gene delivery.
BACKGROUND
The inherent ability of certain peptides or proteins of viral, prokaryotic and eukaryotic origin to bind DNA was used to generate novel peptide-based DNA delivery protocols. We have developed a recombinant approach to make fusion proteins with motifs for DNA-binding ability, Mu and membrane transduction domains, TAT, and tested them for their DNA-binding, uptake and transfection efficiencies. In one of the constructs, the recombinant plasmid was designed to encode the Mu moiety of sequence MRRAHHRRRRASHRRMRGG in-frame with TAT of sequence YGRKKRRQRRR to generate TAT-Mu, while the other two constructs, Mu and Mu-Mu, harbor a single copy or two copies of the Mu moiety.


METHODS
Recombinant his-tag fusion proteins TAT-Mu, Mu and Mu-Mu were purified by overexpression of plasmid constructs using cobalt-based affinity resins. The peptides were characterized for their size and interaction with DNA, complexed with plasmid pCMVbeta-gal, and shown to transfect MCF-7, COS and CHOK-1 cells efficiently.


RESULTS
Recombinant fusion proteins TAT-Mu, Mu and Mu-Mu were cloned and overexpressed in BL21(DE3)pLysS with greater than 95% purity. The molecular weight of TAT-Mu was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to be 11.34 kDa while those of Mu and Mu-Mu were 7.78 and 9.83 kDa, respectively. Live uptake analysis of TAT-Mu, Mu and Mu-Mu as DP (DNA+peptide) or DPL (DNA+peptide+lipid) complexes into MCF-7 cells, followed by immunostaining and laser scanning confocal microscopy, demonstrated that the complexes are internalized very efficiently and localized in the nucleus. DNA:peptide complexes (DP) transfect MCF-7, COS and CHOK-1 cells. The addition of cationic liposomes enhances the uptake of the ternary complexes (DPL) further and also brings about 3-7-fold enhancement in reporter gene expression compared to DP alone.


CONCLUSIONS
Recombinant proteins that are heterologous fusions, having DNA-binding domains and nuclear localization epitopes, generated in this study have considerable potential to facilitate DNA delivery and enhance transfection. The domains in these fusion proteins would be promising in the development of non-viral gene delivery vectors particularly in cells that do not divide.
Rajagopalan
Rukkumani
R
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India.
Xavier
Jennifer
J
Rangaraj
Nandini
N
Rao
Nalam Madhusudhana
NM
Gopal
Vijaya
V
D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D002460:Cell Line; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D006801:Humans; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D015696:Gene Products, tat; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins