Recombinant fusion proteins TAT-Mu, Mu, and Mu-Mu mediate efficient non viral gene delivery

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    THE JOURNAL OF GENE MEDICINE R E S E A R C H A R T I C L EJ Gene Med 2007; 9: 275286.Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/jgm.1014

    Recombinant fusion proteins TAT-Mu, Mu andMu-Mu mediate efficient non-viral gene delivery

    Rukkumani Rajagopalan

    Jennifer Xavier

    Nandini Rangaraj

    Nalam Madhusudhana Rao

    Vijaya Gopal*

    Centre for Cellular and Molecular

    Biology, Uppal Road, Hyderabad

    500007, India

    *Correspondence to: Vijaya Gopal,

    Centre for Cellular and Molecular

    Biology, Uppal Road, Hyderabad

    500007, India.

    E-mail: [email protected]

    Received: 20 October 2006

    Revised: 30 December 2006

    Accepted: 15 January 2007

    Abstract

    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 recombinantapproach 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 pCMV-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 andlocalized 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 37-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. Copyright 2007 John Wiley & Sons, Ltd.

    Keywords gene delivery; non-viral gene delivery; cationic peptides; transfection;cationic liposomes; transfection; TAT-Mu; recombinant DNA; gene expression

    Copyright 2007 John Wiley & Sons, Ltd.

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    276 R. Rajagopalan et al.

    Introduction

    The natural process of DNA uptake by eukaryotic cells

    is inefficient owing to the chemical nature of the gene

    and the cell membrane. Invariably, gene delivery has to

    be facilitated by carriers both viral and non-viral withexamples of cationic lipids [1,2] and polymers [3,4] in

    the non-viral category. These are known to interact with

    DNA resulting in the formation of condensed, transfection-

    competent complexes. Gene delivery by non-viral methods

    is less efficient in cells where the mitotic rates are low.

    Cells that divide slowly or differentiated cells are often

    resistant to current DNA delivery methods. Understand-

    ing the mechanism of nucleic acid uptake by cells coupled

    with the development of methods to make this an efficient

    process forms the major activity of research in the area of

    gene delivery. Although gene transfer methods based on

    viruses are successful, challenges such as immunogenicity

    [5], toxicity [6] and their ability to integrate into the hostgenome [7] have to be overcome. Cationic amphiphiles

    have been widely employed for in vitro transfections as

    these offer many advantages over that of viral systems in

    terms of the ease of production, ability to carry larger DNA

    transgenes, stability and safety in clinical trials [2,8]. In

    the past decade, the optimization of conditions for trans-

    fection has taken a rather long route as a large number

    of these non-viral delivery agents and their derivatives

    are initially tested empirically for their efficiency. A more

    rational approach is to address the issues concerning vari-

    ous barriers and pathways leading eventually to the entry

    of DNA or transfection complexes of DNA into the nucleus,which is the ultimate barrier in the transfection process.

    This is elegantly discussed in recent reviews pertaining to

    intracellular trafficking of nucleic acids, non-viral vectors

    and polyplexes [9,10] that forms a major part of non-viral

    gene delivery strategies.

    Cationic peptides that condense DNA have a tremen-

    dous potential to facilitate the passage of DNA through

    various mechanisms [1117] thereby circumventing

    cell membrane barriers. The incorporation of pep-

    tides in transfection protocols with DNA-binding poten-

    tial/nuclear localization motifs has been shown to

    improve the transfection efficiency [14,15,1820] and

    may offer solutions to deliver DNA more effectively.Several adenoviral proteins such as the Mu, with pre-

    dominantly basic amino acid residues, have the potential

    to bind DNA and also bring about enhancement in cationic

    lipid-mediated transfection [13,19]. Mu () is based on

    the adenoviral core peptide of 19 amino acids with a

    sequence MRRAHHRRRRASHRRMRGG, twelve of which

    are basic, conferring positive charge on the protein. This

    peptide is found associated with the adenoviral core com-

    plex [21,22]. Work in Millers laboratory has shed light

    on using systems that are based on the DNA-condensing

    ability of synthetic Mu peptide and has also shown its

    ability to enhance cationic lipid-mediated transfectionsin vitro [13].

    The movement of macromolecules within cells is often

    aided by nuclear localization sequences and factors that

    recognize and bind them. The use of such sequences in the

    preparation of transfection complexes may therefore facil-

    itate transport of the complexes efficiently to the nucleus

    where gene expression must eventually occur [23], which

    is the major rate-limiting step [10,24]. Transfection-

    enhancing peptides, synthesized by solid-phase synthesis,

    have reportedly shown reporter gene expression in various

    cell lines.

    Our strategy in this study aims at designing and pro-

    ducing small proteins encoded by plasmid constructs by

    conventional recombinant DNA strategies and bacterial

    fermentation rather than the synthetic route. A basic

    domain of TAT was earlier shown to be the minimal

    sequence responsible for the cellular and nuclear uptake

    which is contributed by potential nuclear localization

    sequences (NLSs) in the 11-amino acid epitope YGRKKR-

    RQRRR [25]. The TAT moiety has also been shown to

    deliver functional fusion proteins in vivo [26]. By com-

    bining the Mu motif with TAT, the inherent, desiredproperties, such as DNA condensation of Mu and nuclear

    localization conferred by TAT, facilitation of transport

    across plasma and nuclear membrane, could be integrated

    into one molecule. Here, we report the generation of the

    plasmid constructs that overproduce TAT-Mu, Mu, and

    Mu-Mu as heterologous, his-tag fusion proteins. We eluci-

    date their DNA-binding, uptake and transfection potential

    into cells. Recombinant fusion proteins TAT-Mu, Mu and

    Mu-Mu have the potential to bind plasmid DNA, increase

    internalization of transfection complexes into adherent

    cells in culture, and thereby enhance transfection.

    Materials and methods

    Chemicals

    1,2-Dioleoyl-3-trimethylammonium propane (DOTAP),

    3 beta-(N(N,N-dimethylaminoethane)carbamoyl) choles-

    terol (DC-Chol) and dioleoylphosphatidylethanolamine

    (DOPE) were purchased from Avanti Polar Lipids Inc.

    N-(Lissamine rhodamine B sulfonyl)-1,2-dihexadecanoyl-

    sn-glycero-3-phosphoethanolamine (Rh-DHPE) was from

    Molecular Probes. Plasmid pTAT was a kind gift from

    Steve Dowdys laboratory (UCSD). Plasmid pHind2 wasa kind gift from Akusjarvi of Uppsala University Sweden.

    Plasmid DNA pCMV.SPORT--gal was from Invitrogen.

    BD TALON was from BD Biosciences. The primary 6xhis

    monoclonal antibody was from BD Biosciences. FITC

    secondary antibodies were from Bangalore Genei. Flu-

    orescein DNA-labeling kits for labeling plasmid DNA by

    nick translation and random primer labeling were from

    Jonaki, India. Ethidium bromide (EtBr) was from Sigma

    Aldrich. All other chemicals used were of the highest

    purity available.

    PCR amplification, sub-cloning

    The Mu moiety from the plasmid pHind2 was amplified

    using forward and reverse primers with restriction

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    Recombinant Fusion Proteins Mediate Efficient Gene Delivery 277

    sites KpnI and EcoRI incorporated in them. This was

    subsequently cloned in-frame into the pTAT construct

    similarly restricted to generate pTAT-Mu that is inducible

    in the presence of 1 mM IPTG when expressed in

    an overexpression host BL21(DE3)pLysS. In the Mu

    construct, a BamH I digestion of pTAT-Mu followed by

    recircularization has deleted the TAT moiety. Inserting a

    second copy of Mu amplified by polymerase chain reaction

    (PCR) into the Mu construct generated the Mu-Mu

    plasmid. These constructs, pTAT-Mu, pMu and pMu-Mu,

    were verified for the integrity of the DNA insert sequence

    prior to transformation in E. coli. The plasmids were then

    transformed into competent E. coli BL21(DE3)pLysS cells,

    grown and induced to express the desired protein.

    Overexpression and purification of therecombinant proteins

    Recombinant TAT-Mu, Mu and Mu-Mu proteins were

    purified from bacterial pellets by urea denaturation fol-

    lowed by the use of cobalt affinity matrix (BD TALON;

    BD Biosciences). This matrix eliminates the binding of

    endogenous contaminating E. coli histidine-rich protein

    that co-purifies with our protein in a nickel-affinity matrix,

    thereby enriching the protein of our interest during elu-

    tion. PD-10 desalting columns subsequently removed urea

    employed as a denaturant in the purification protocols.

    The final yield was typically 2 mg from a 1000 ml culture

    where the protein was greater than 95% homogeneous,

    as ascertained by sodium dodecyl sulfate/polyacrylamide

    gel electrophoresis (SDS-PAGE). Western blotting of thesepurified proteins was carried out using monoclonal anti-

    his antibodies against the 6xhis-tag fusion proteins fol-

    lowed by binding to the secondary antibody.

    DNA binding and electrophoreticmobility shift assays

    Charge neutralization and DNA condensation of the

    fusion peptides were observed by electrophoresis of

    the complexes on agarose gels. Plasmid DNA pEGFPN3(0.20.4 g) was complexed with increasing amounts

    of the TAT-Mu/Mu/Mu-Mu peptide, to obtain the corre-

    sponding charge ratios, in Hepes buffered saline (150 mM

    NaCl, 5 mM KCl, 0.75 mM Na2HPO4, 20 mM Hepes pH

    7.4) and incubated for 25 min at room temperature (RT).

    The complexes were subsequently analyzed on a 1%

    agarose gel [Tris-acetate buffer (TAE) system, pH 8.2]

    and stained with EtBr post-electrophoresis.

    EtBr exclusion assay

    The binding of DNA with TAT-Mu, Mu and Mu-Mu was

    studied by monitoring the fluorescence with the fluores-cent probe EtBr. The intercalation of EtBr into DNA brings

    about an increase in thefluorescence quantum yield. Upon

    binding and condensing DNA, EtBr is expelled from the

    DNA-EtBr complex and this displacement of EtBr by the

    fusion proteins is reflected as a drop in the fluorescence

    signal. EtBr from a stock solution of 1 mg/ml was added to

    DNA in a cuvette and the fluorescence was measured using

    a fluorescence spectrophotometer (Hitachi-F4000). The

    excitation wavelength, ex

    , was 516 nm and the emission

    wavelength was kept at 598 nm (slit width 5 5 nm).

    Briefly, 2.3 g of pCMV--gal plasmid DNA was added to

    500 l of sample in 20 mM Tris-HCl buffer (pH 7.4) in

    a fluorescence cuvette. EtBr (0.23 mg) was added to the

    DNA solution and the baseline fluorescence was deter-

    mined. The fluorescence intensity obtained upon each

    addition was normalized relative to the fluorescence sig-

    nal of the DNA-EtBr complex in the absence of the fusion

    proteins, which was taken as 100%. The binding of DNA

    by the fusion peptides TAT-Mu, Mu and Mu-Mu, respec-

    tively, was recorded after each addition at time intervals

    of 5 min. The binding efficiency correlates with a drop in

    the fluorescence intensity at various time points.

    Preparation of complexes fortransfection

    Cationic liposomes DOTAP and DC-Chol were prepared

    by drying the appropriate amount from their chloroform

    stock solutions. Plasmid DNA pCMV-gal was purified by

    using the Qiagen kit (endotoxin free) using the manu-

    facturers protocol. Complexes were prepared by taking

    0.9 g of plasmid DNA and varying amounts of the fusion

    protein to obtain the desired charge ratio, in Hepesbuffered saline (pH 7.4), and incubated for 25 min at

    RT. Transfection experiments were initially performed

    with the TAT-Mu, Mu and Mu-Mu peptides and DNA

    complexes. In liposome-mediated transfections, cationic

    liposomes were added to the pre-incubated DNA : peptide

    complexes and further incubated for 25 min at RT. This

    ternary complex was then diluted with serum-free Dul-

    beccos modified Eagles medium (DMEM) to a final

    volume of 300l before splitting them into triplicates

    of 100 l volume on a 96-well plate format containing

    20 000 cells per well. The cells were incubated for at least

    3 h following which complete medium with serum was

    added and the reporter gene expression was assayed for-galactosidase 2448 h post-transfection.

    Live uptake and immunolocalization ofpeptide : DNA complexes by confocalmicroscopy

    MCF-7 cells were grown on either a chambered cover

    glass (Lab-Tek, Nalgene, Nunc International Corp.) or

    on cover slips placed on six-well plates. Cells plated to

    70% confluence were washed with serum-free DMEM

    (pH 7.4) and then incubated with the complexes at 1 : 4DNA : peptide charge ratio, at 37 C and 5% CO2. In the

    case of fluorescent cationic lipid, DOTAP was labeled

    with 5 mol% Rh-DHPE. For the live uptake experiments,

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    278 R. Rajagopalan et al.

    plasmid DNA was labeled (at 1 : 1 ratio of labeled vs.

    unlabeled DNA) by either the random primer labeling

    kit or by the nick translation kit using fluorescein-d UTP

    from Jonaki, combined with the peptide and incubated

    at 37 C. Subsequently, these were examined by confocal

    microscopy to monitor the movement of the complexes

    into cells. The image acquisition conditions for all the

    experiments were identical.

    The immunolocalization experiment was similar to the

    live uptake experiment except that, after the incuba-

    tion process, cells were exposed to anti-his antibodies

    directed against the 6xhis-tag fusion proteins after uptake.

    After incubation for 1 h, the cells were fixed with 2%

    paraformaldehyde, permeabilized with 0.25% Triton X-

    100 followed by immunostaining with anti-his antibodies,

    at a dilution of 1 : 200, and incubated overnight at RT.

    This was followed by the addition of propidium iodide

    (PI) with the secondary antibody FITC-conjugated goat

    anti-mouse IgG (anti-mouse used at a dilution o f 1 : 1000and then analyzed with a confocal microscope (LSM 510

    META, Carl Zeiss). Optical sections of cells were taken

    using a 63 objective at 0.5 m intervals. A 518 nm

    laser line was used to excite the FITC fluorophore in the

    secondary antibody and a 543 nm laser to visualize the

    rhodamine fluorophore. Images were collected simulta-

    neously for PI staining of the nuclei. The images were

    analyzed by taking equal numbers of optical sections in

    the central region and then merged.

    Results

    Construction and purification ofmultifunctional DNA carrier proteinsTAT-Mu, Mu and Mu-Mu

    The DNA-binding domain of Mu was derived as described

    in the Materials and methods section by restriction

    digestion, PCR amplification and assembly in-frame in

    the expression construct pTAT to obtain pTAT-Mu. The

    pMu construct was obtained by BamH I excision of the

    TAT moiety of pTAT-Mu plasmid. In the case of Mu-

    Mu, an additional copy of the Mu moiety was clonedin-frame into the pMu construct. All the three plasmid

    constructs encoding the fusion protein are under the con-

    trol of the T7 promoter with an N-terminal histidine tag.

    Figure 1A shows the schematic of the fusion constructs

    with the placement of 6xhis-tag at the N-terminus. The

    presence of the his-tag in-frame with TAT-Mu, Mu and

    Mu-Mu facilitates the purification of the recombinant pro-

    teins using cobalt affinity matrix. The fusion proteins

    were expressed in E. coli BL21(DE3)pLysS. Total bacterial

    lysates were obtained with 8 M urea and the recombi-

    nant protein purified by elution with 250 mM imidazole.

    The fractions were analyzed on 15% SDS gels, pooledand then the denaturant and imidazole were removed

    with PD-10 columns. Figure 1B shows an SDS-PAGE anal-

    ysis of the purified proteins appearing as single bands

    with an apparent molecular weight that is slightly larger

    than that deduced, based on the DNA sequence of the

    plasmid inserts. The recombinant TAT fusion proteins

    migrated slower than expected with an apparent increase

    of 710 kDa on SDS gels. This apparent difference could

    be attributed to both the TAT leader sequences as well

    as the highly cationic nature of the protein. That the

    TAT leader sequences contribute to 5 kDa increase in

    molecular weight has been reported for other TAT fusion

    proteins [27]. The presence of the his-tag of the fusion

    proteins was confirmed by immunoblotting using anti-his

    antibodies that confirmed the authenticity of the proteins

    (inset of Figure 1B, lanes 13). The corresponding molec-

    ular masses for TAT-Mu, Mu and Mu-Mu, as derived by

    matrix-assisted laser desorption/ionization time-of-flight

    mass spectrometry (MALDI-TOFMS), are 11.34, 7.78 and

    9.83 kDa, respectively.

    DNA-binding analysis

    Gel retardation assay

    We next investigated the ability of recombinant proteins

    TAT-Mu, Mu and Mu-Mu to bind plasmid DNA by an

    agarose gel retardation assay. Plasmid pEGFPN3, harbor-

    ing thegene encoding thegreen fluorescent protein (GFP),

    was used for the binding assay. The number of arginines

    and lysines in the fusion protein were taken into account

    in order to calculate the total positive charges on the

    molecule. Complexes of DNA and protein were prepared

    at various charge ratios and incubated with the plasmidat 37 C for 15 min and electrophoresed. In the absence

    of protein, which is the control, both relaxed and super-

    coiled DNA migrated normally. At charge ratios of 1 : 8

    and 1 : 16 (DNA: protein), the ability to bind DNA was

    the highest for Mu-Mu while both TAT-Mu and Mu had

    similar binding patterns, as seen from the shift in the

    mobility of plasmid DNA. Both relaxed and supercoiled

    forms of DNA were retarded by their interaction with

    the fusion proteins (Figure 1C, panel a TAT-Mu; panel

    b Mu; panel c Mu-Mu). In the case of Mu-Mu at 1 : 8

    charge ratio, the binding to EtBr is occluded as seen from

    the decreased DNA band intensity. At 1 : 16 charge ratio,

    the DNA appears to be inaccessible to EtBr binding. The

    ability of the peptides to protect DNA in the complex from

    nucleases was investigated by incubating the complexes

    with DNase I. Protection from nucleases was complete at

    charge ratios of 1 : 8 (data not shown).

    Displacement of EtBr from DNA by TAT-Mu, Mu and

    Mu-Mu: DNA-binding assay

    Titrating DNA-EtBr complexes with the fusion proteins

    further elucidated the differential DNA-binding charac-

    teristics. In this experiment, the addition of the fusion

    proteins to the DNA-EtBr complexes resulted in a rapiddecrease in the fluorescence intensity (Figure 1D). The

    binding efficiency of the fusion proteins correlates with a

    drop in the fluorescence intensity and this loss could be

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    Recombinant Fusion Proteins Mediate Efficient Gene Delivery 279

    interpreted as their ability to bind DNA that results in the

    displacement of the intercalated EtBr. Although the three

    fusion proteins were able to bind DNA, as seen from the

    gel binding assays depicted in Figure 1C, the fluorescence

    titrations clearly indicate that the efficiency with which

    the DNA is condensed appears to be different in the case

    of Mu-Mu, which is the most efficient, while TAT-Mu

    and Mu displayed similar DNA-binding affinity. When we

    monitored the binding properties of TAT, we observed

    that TAT does not have any effect on EtBr exclusion even

    at 1 : 8 charge ratio indicating its lack of binding to DNA

    (not shown).

    TAT-Mu, Mu and Mu-Mu mediatetransfer of DNA into cells

    Live uptake

    Having ascertained the DNA-binding characteristics of the

    fusion proteins, the internalization of DNA : peptide (DP)

    or DNA : peptide : lipid (DPL) complexes into the cells

    was investigated by monitoring the live uptake of the

    complexes by confocal laser scanning microscopy. After

    the application of complexes of DP or DPL, cells wereincubated in the absence of serum for 1 h and replaced

    with Hepes/Hanks buffer containing 10% serum. In the

    case of the DP complexes, nick translated plasmid DNA

    was combined with the fusion proteins and the uptake

    into MCF-7 cells was visualized live. The localization of

    DP complexes appeared as punctate particles in both the

    cytoplasm and the nucleus. It is evident that the DP

    complexes, D + TM, D+ Mu and D + Mu-Mu, prepared

    with TAT-Mu, Mu and Mu-Mu, respectively, facilitate the

    Figure 1. Construction and gel analysis of DNA-binding carrier

    proteins. (A) Schematic representation of chimeric fusion

    constructs encoding (a) TAT-Mu, (b) Mu, and (c) Mu-Mu. Themotifs harboring the TAT sequence YGRKKRRQRRR and/or Mu

    sequence MRRAHHRRRRASHRRNRGG are cloned in-frame in a

    pTAT expression vector and expressed as N-terminal his-tag

    fusion proteins in E. coli. The plasmids are designated as

    pTAT-Mu, pMu and pMu-Mu. The Mu construct is created by

    a deletion of the TAT moiety of the plasmid construct pTAT-Mu

    as described in the Materials and methods section. (B) Sodium

    dodecyl sulfate/polyacrylamide gel electrophoretic (SDS-PAGE)

    analysis of the purified fusion proteins. Lane 1, 2, 3 correspond

    to TAT-Mu (2g),Mu (2 g) and Mu-Mu (1 g) after desalting

    from PD-10 columns. The molecular weights of the expressed

    proteins TAT-Mu, Mu and Mu-Mu are 11.34, 7.78 and 9.23 kDa,

    respectively. Inset shows the immunoblot analysis of purified

    proteins TAT-Mu, Mu and Mu-Mu after electrophoresis and

    Western transfer. The 6xhis-tagged proteins were detected withmonoclonal anti-his antibodies followed by secondary antibody

    goat anti-mouse IgG AP conjugate. Detection was by NBT/BCIP

    treatment. M, molecular weight standards in kDa. (C) Gel

    retardation assay of the fusion proteins TAT-Mu, Mu and Mu-Mu.

    In each panel, i.e. (a)(c), plasmid pEGFPN3 (0.4 g in (a) and

    0.2 g in (b) and (c)) was incubated in 1 Hepes buffered

    saline (pH 7.4) with increasing amounts of the purified protein

    corresponding to the charge ratio indicated above each lane.

    Samples were electrophoresed on 1% agarose gel in TAE buffer

    and visualized by staining with ethidium bromide (EtBr). Control

    in each panel pertains to plasmid DNA alone. (D) DNA binding

    of TAT-Mu, Mu and Mu-Mu to DNA: Titration curves depicting

    the release of EtBr from pCMV-gal plasmid DNA upon binding

    TAT-Mu (solid squares), Mu (solid triangles) and Mu-Mu (open

    circles) fusion proteins in a buffer containing 10 mM TrisHCl(pH 7.4). 8 g of protein corresponds to approximately 1 : 2.5

    charge ratio (DNA : protein). Details as in the Materials and

    methods section

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    280 R. Rajagopalan et al.

    Figure 2. Live uptake of transfection-competent complexes into MCF-7 cells. (A) Confocal microscope analysis of uptake of

    FITC-labeled DNA+ fusion protein complexes into MCF-7 cells. Panels from left to right: Control: DNA alone; DNA : TAT-Mu;

    DNA: Mu; DNA : Mu-Mu all at 1 : 4 charge ratio DNA: protein. 0.9 g of DNA was used to make the transfection complexes with

    the corresponding protein and applied to cells in the absence of serum. Uptake of the complexes is for a duration of 3 h before

    replacement with complete medium containing 10% serum. Thirty minutes before the confocal analyses, the cover slips were placed

    in Hepes/Hanks buffer. (B) Confocal microscope analysis of uptake of DNA+ fusion protein+ lipid complexes into MCF-7 cells.

    Complexeswere prepared using rhodamine-labeled DOTAP liposomes (5 mol% Rh-DHPE) and the uptake experiment was carried out

    as described in the Materials and methods section. Panels on the left show the images in the fluorescence channel and those on the

    right show the images merged with the transmission channel. Eight optical sections in the middle region, each of 0.5 m thickness,

    were combined to generate the images. D+ L = DNA:liposomes at 1 : 1 charge ratio, D+ TM+ L = DNA+ TAT-Mu + liposomes(1: 4: 1), D+Mu+ L = DNA+Mu+ liposomes (1 : 4 : 1 charge ratio) and D+Mu-Mu + L = DNA+Mu-Mu+ liposomes (1 : 4 : 1)

    uptake of DNA into the cytoplasm and the nucleus when

    compared to the control DNA (extreme panel on the

    left) that is labeled DNA alone (Figure 2A). Both nick

    translation and random primer labeling methods to label

    plasmid DNA yielded identical end results.

    In the case of live uptake of DNA : peptide : lipid

    complexes (DPL), cationic lipid DOTAP was labeled with

    5 mol% Rh-DHPE. The punctate red fluorescence (seen

    in all panels) was observed upon uptake and entry

    of the particles into the cytoplasm. When comparedto DL (DNA+ lipid) complexes, the uptake of DPL

    complexes prepared with the three fusion proteins

    was enhanced as seen from the increased fluorescence

    intensity (Figure 2B). In a situation where both labeled

    DNA and rhodamine-labeled lipid were used to prepare

    the DPL complexes, with the respective proteins, the

    presence of a dual label, originating from the FITC-labeled

    plasmid DNA as well as the rhodamine-labeled lipid,

    within cells indicated that the DNA and lipid co-localized

    (not shown).

    Immunocytochemistry of TAT-Mu, Mu and Mu-Mu fusion

    proteinsIn yet another set of experiments, we also monitored

    the intracellular localization of the complexes after an

    incubation period of 1 h followed by immunolocalization

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    Recombinant Fusion Proteins Mediate Efficient Gene Delivery 281

    Figure 3. Immunolocalization and confocal microscopy of fusion proteins. Fusion proteins : plasmid pCMV-gal DNA complexes

    (DP) prepared with TAT-Mu (a), Mu (b), and Mu-Mu (c) at 1 : 4 charge ratio and incubated for 1 h with cells at 37 C. After

    incubation, cells were fixed followed by immunostaining with anti-his antibodies followed by propidium iodide (PI) addition with

    the FITC-conjugated secondary antibody goat anti-mouse IgG. The localization of the his-tagged proteins in the cytoplasm and the

    nucleus is visualized as green fluorescence while PI staining of the nucleus is in red. Equal numbers of optical sections in the

    middle region, each of 0.5 m thickness, were combined to generate panels in (a)(f). Top panels correspond to DP complexes

    alone incubated with MCF-7 cells for 1 h while the bottom panels (df) correspond to TAT-Mu, Mu and Mu-Mu, respectively, to

    which DPL complexes prepared with DC-Chol: DOPE at 1 : 4:1 charge ratios were applied to cells and incubated for 1 h. Details as

    described in the Materials and methods section

    with FITC-labeled secondary antibodies (Figure 3). We

    observed that the entry of complexes into the cytoplasm

    and nucleus occurs rapidly within 1 h, as seen from the

    green fluorescence in the cytoplasm and the nucleus

    in the case of DP complexes prepared with TAT-Mu,

    Mu or Mu-Mu (Figures 3a3c). The nuclear staining is

    very intense in all the panels where cells were treated

    with DPL complexes prepared with DC-Chol : DOPE

    (Figures 3d 3f). Panels a and b in the Supplemental

    Section S1 correspond to without-primary antibody

    control and cell control, respectively (see Supplementary

    Material).

    TAT-Mu, Mu and Mu-Mu transfection in MCF-7 cells

    Having ensured the DNA-binding and uptake propertiesof DP and DPL complexes prepared with the recombinant

    fusion proteins TAT-Mu, Mu and Mu-Mu, we went on

    to determine the relative transfection efficiencies of

    fusion proteins. Transient transfections during the initial

    characterization of the fusion proteins TAT-Mu, Mu and

    Mu-Mu were tested at various charge ratios varying from

    1 : 2 to 1 : 16. We have consistently obtained maximum

    transfection when cells were transfected with complexes

    at 1 : 8 charge ratio of DNA : fusion protein with TAT-Mu,

    Mu or Mu-Mu (not shown).

    After the initial characterization, transient transfections

    were carried out in MCF-7 cells with plasmid pCMV-gal : lipid DL or DPL complexes prepared with TAT-

    Mu, Mu or Mu-Mu fusion proteins and reporter gene

    expression was assayed 48 h post-transfection, as detailed

    in the Materials and methods section. We compared

    the transfection efficiency of the transfection complex

    at the optimized charge ratio of 1 : 8. We chose the

    optimized charge ratio of 1 : 1 DNA : lipid as in our

    hands DDAB : DOPE, Lipofectin and also DC.Chol : DOPE

    gave the best results. Where complexes (DPL) were

    prepared with the fusion proteins and the cationic lipid,

    we obtained enhanced transfection at 1 : 8:1 charge

    ratio of D : P : L. Figures 4a4c show a representative

    set of the results obtained in MCF-7 cells. Complexes

    of pCMV-gal : fusion protein DP, i.e. D+ TM, D + Mu or

    D + Mu-Mu, at 1 : 8 charge ratio, were very effective at

    transfecting cells when compared to plasmid pCMV-gal

    alone. Lipopolyplex mixtures DPL prepared with TAT-

    Mu (D+ TM+ L) resulted in 7.5-fold enhancement

    in transfection when compared to D + TM and 3.5-fold increase was observed when compared to D + L

    (Figure 4a). Similar fold increases were obtained for TAT-

    Mu with the cationic lipid Lipofectin or DDAB : DOPE (not

    shown).

    In the case of the fusion protein Mu, 4-fold

    increase in the transfection efficiency with D + Mu+ L

    (L = DC-Chol : DOPE) was obtained when compared with

    D + Mu complexes. The fold increase in transfection

    efficiency obtained with D + Mu+ L prepared with DC-

    Chol: DOPE is 3-fold when compared with that of

    D + L (Figure 4b). We also obtained good transfection

    efficiencies with Mu-Mu fusion protein where D+

    Mu-Mu+ L complexes resulted in 7-fold enhancement

    in transfection when compared to D + Mu-Mu alone

    and 3-fold when compared with D + L. Complexes

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    282 R. Rajagopalan et al.

    Figure 4. Transient transfection of MCF-7 cells with (a) TAT-Mu(TM), (b) Mu, and (c) Mu-Mu. Complexes prepared with the

    fusion proteins and the lipids are at a charge ratio of 1 : 8:1 and

    prepared as indicated in the Materials and methods section.

    Protein estimation of the cell lysates was done to express

    -galactosidase activity in mU after normalizing the protein

    values. D-plasmid DNA alone (control); L-lipid DC-Chol : DOPE.

    The lipids used were DC-Chol : DOPE in all panels at 1 : 1

    charge ratio. The reporter gene assay was carried out 48 h

    post-transfection

    of the fusion proteins and plasmid DNA harboring the

    -galactosidase or GFP gene also transfect COS, HeLa

    and CHOK1 cells efficiently both in the absence andpresence of cationic liposomes (data not shown). Purified

    TAT as such did not result in any enhancement in

    transfection either in the absence or presence of Lipofectin

    or DC.Chol : DOPE under the conditions tested (not

    shown).

    A charge ratio of 1 : 1 of DNA to lipid was generally

    found to be optimal for several cationic lipids developed

    in our laboratory and with those reported in the literature.

    Unlike cationic lipids, higher charge ratios greater than

    1 : 8, resulting from peptides in a polyplex, were non-toxic.

    The effect of charge ratio in a lipoplex in transfection is

    not similar. In the case of DPL complexes, a charge ratio

    higher than 1 : 8:1 or lower than 1 : 4:1 always resulted in

    less efficient transfection (not shown).

    We also examined the transfection efficiency of a

    control peptide such as poly-l-lysine in the presence and

    absence of Lipofectin. Poly-l-lysine 3 kDa and 30 kDa

    were both tested in MCF-7 cells and compared to a lipid-

    DNA control under the same conditions. The transfection

    efficiency with poly-l-lysine in the absence of lipid was

    similar to the fusion peptides. The transfection efficiency

    with the three fusion proteins was 58 times higher when compared to poly-l-lysine in the presence of the

    lipid (not shown).

    Discussion

    Non-viral vectors are comparatively much safer although

    not as efficient as viral vectors [8]. It is vital to

    develop non-viral vector systems and make them clinically

    efficient. The use of peptides to overcome the cellular

    barriers of DNA entry into the cell and then into the

    nucleus is gaining momentum. When testing out gene

    delivery systems, the cell membrane is the first major

    barrier. An approach to overcome this problem is to

    incorporate motifs with peptides that are capable of

    penetrating the cell membrane. Subsequently, the delivery

    of DNA from the cytoplasm into the nucleus, that is also an

    inefficient process and considered to be the major barrier

    in non-dividing cells, is challenging. A strategy to improve

    nuclear uptake of DNA is to exploit the cellular nuclear

    import machinery where peptides containing an NLS can

    be conjugated and thereby facilitate the translocation

    of macromolecules across the nuclear envelope inside

    cells. Few synthetic peptides with DNA-binding ability ornuclear import capabilities have been shown to enhance

    transfection efficiency. In this report we have presented a

    strategy to make combinations of various peptide motifs

    using recombinant genetic approaches and to produce

    them by heterologous expression. The approach allows

    the testing of various combinations of the peptides that

    are efficient DNA carriers. Based on this, novel peptide-

    based gene carriers could be designed for gene therapy

    applications.

    Our efforts to identify potential cationic proteins for

    transfection led to the construction of plasmids that

    encode the fusion proteins TAT-Mu, Mu and Mu-Mu thatarecationic in nature. Mu is characterized by the 19-amino

    acid epitope, rich in arginines, and potential to condense

    the viral DNA [21]. While Mu helps condense DNA,

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    Recombinant Fusion Proteins Mediate Efficient Gene Delivery 283

    the short, basic peptide sequence from the HIV-1 TAT

    protein known as TAT or protein transduction domain

    (PTD) (residue 4757) confers the property of cellular

    uptake across membranes and nuclear localization. As

    evident from our results, the single and double copies of

    the Mu moiety in the recombinant Mu and Mu-Mu are

    as efficient as TAT-Mu in bringing about reporter gene

    expression although the DNA-binding pattern of Mu-Mu

    is different from that of TAT-Mu and Mu. Interestingly

    the transfection efficiency of these DP complexes was

    further enhanced when the transfection complexes were

    formulated with cationic liposomes, i.e. DPL. These fusion

    proteins have enhanced the transfection of efficient

    and well-studied cationic lipid formulations of which

    only results obtained with DC-Chol : DOPE have been

    presented here.

    Although several factors such as the primary sequence,

    net charge and sequence context may all be important,

    a combination of these could possibly influence genetransfer in a positive manner. The presence of the TAT

    motif in TAT-Mu may thereby synergistically facilitate

    the import of plasmid DNA into the nucleus, as it

    has better DNA-binding ability than TAT alone. These

    properties finally relate to the enhanced transfection

    observed for TAT-Mu both in the absence and presence

    of cationic lipids. Hyndman et al. have observed the

    unexpected negative effect of also tagging TAT with

    poly-l-lysine [19] which indicates that the primary

    sequence of TAT may play a role in determining peptide-

    mediated enhancements in the transfection efficiency in

    the presence of liposomes. From the results with poly-l-lysine, we reasoned that a stretch of positive charges

    in the cationic polymer may not be as efficient as

    peptides described in this study. The presence of multiple

    arginines in the double copy of the Mu-Mu construct

    may also explain the stronger binding to DNA as seen by

    immunolocalization. Nuclear import of nucleic acids may

    be inefficient during cationic lipid-mediated transfection.

    A number of attempts to improve the transfection

    efficiency by attempting to enhance nuclear uptake of

    nucleic acids by using polycationic polymers bearing

    peptide sequences with nuclear localizing capabilities

    have met with success. The presence of NLSs does

    not promote transfection without adequate nucleic acidcharge neutralization and condensation as well. For

    example, synthetic TAT alone complexed with DNA was

    unable to transfect cells but facilitated transfection in

    the presence of cationic liposomes [19]. The domains in

    TAT-Mu provide the attributes important in contributing

    the individual functions and may lay the foundation for

    designing next-generation constructs where issues such

    as the binding strength and nuclear localization potential

    need to be considered. In the early studies, with cationic

    peptides in transfection, the peptides were generated by

    solid-phase synthesis. Such labor-intensive methods of

    peptide synthesis limit testing of larger peptides and alsopeptide combinations. To circumvent these issues, few

    laboratories have attempted to synthesize peptides using

    recombinant DNA approaches. Heterologous production

    of peptides rich in basic amino acids, in bacterial systems,

    usually results in low yields and in our hands required

    the standardization of protocols to further improve

    the yields. In our experience, the fusion proteins are

    present as insoluble fractions and require denaturation

    followed by renaturation to obtain pure preparations.

    To aid in purification of the peptides, DNA sequences

    corresponding to the peptides were cloned downstream

    of the his-tag present at the N-terminus. The presence of

    the his-tag at the N-terminus enabled us to confirm the

    purity of the peptide preparation by Western blotting.

    In addition, the presence of histidine residues in a

    transfection complex is known to bring about osmotic lysis

    of endosomes upon protonation due to acidification of

    endosomes [28] conferring a broad utility of these fusions

    in gene therapy applications. All the three fusion proteins,

    TAT-Mu, Mu and Mu-Mu, have DNA-binding properties

    and also the ability to protect the DNA from nucleases. The

    charge ratio required for retardation of the plasmid DNAand protection from nuclease occurs at 1 : 8 DNA : protein,

    indicating complete occlusion of the plasmid by peptides.

    Within 1 h of addition of DP, extensive internalization of

    peptides along with the plasmid DNA was observed. The

    rapid entry of DP and particulate fluorescence suggests

    that the endocytic pathway mediates the uptake of the

    complexes. The cellular entry of DP or DPL complexes

    was confirmed by using fluorescent probes where both

    DNA and the lipid were shown to co-localize. Based on

    intensity of fluorescence as shown by immunostaining and

    a fluorescence-based DNA binding assay, it is apparent

    that Mu-Mu shows maximum internalization even in theabsence of lipid. A significant amount of fluorescence was

    also seen inside the nucleus suggesting that in 1 h DP or

    DPL complexes have entered the nucleus. In comparison

    to this, Mu apparently shows more internal fluorescence.

    When the internal fluorescence was monitored using the

    his-tag antibodies, the results showed much stronger

    fluorescence inside the nucleus of MCF-7 cells. The

    intensity was more pronounced with DPL than DP. Though

    significant particulate fluorescence is seen with DP or

    DPL in fixed samples strong fluorescence seen inside the

    nucleus may be partially attributed to the process of

    fixation per se. During fixation, the cationic peptides may

    access the nuclear DNA and bind avidly thereby resultingin strong fluorescence. However, the results obtained

    with live imaging of DP uptake using FITC-labeled DNA

    and co-localization experiments clearly indicated that

    these peptides have facilitated rapid entry of the DP

    or DPL into the MCF-7 cells. Efficient internalization of

    plasmid DNA was supported by our data that the fusion

    proteins are able to bring about reporter gene expression

    in MCF-7 cells (100-fold) when compared to plasmid

    pCMV-gal DNA alone. Between the three fusions, the

    efficiency of transfection was enhanced in the presence

    of DC-Chol : DOPE contributing to 3 7-fold differences

    between DP and DPL for TAT-Mu, Mu and Mu-Mufusion proteins. It has been demonstrated that cationic

    polymers such as poly-l-lysine and protamine enhance

    lipid-mediated transfections in several cell lines in vitro

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    284 R. Rajagopalan et al.

    [29]. Similarly, several DNA-condensing natural peptide

    sequences including Mu [13], HMG [30], and histones

    [31], etc., have been demonstrated to enhance reporter

    gene expression when included in the transfection

    protocols. A recent study from Wels laboratory has shown

    that recombinant derivatives of human HMGB2 facilitate

    non-viral gene delivery [15]. Yet another study used a

    similar approach by generating a recombinant polymer-

    fusion protein (KH)-FGF2 that had the ability to condense

    DNA and mediate transgene expression in mammalian

    cells [32].

    Synthetic Mu was extensively investigated for its ability

    to enhance transfection efficiency in post-mitotic neuronal

    cells and also in vivo lung transfection. These studies

    have led to LMD formulations containing lipids and Mu

    peptide which are efficient transfection formulations at

    very low amounts of DNA and in the presence of 100%

    serum [13]. Examination of peptide sequences of the

    fusion proteins with PSORT II [33] predicts the nuclearlocalization scores of the TAT-Mu, Mu and Mu-Mu to be

    similar. The contribution of arginines as a key feature in

    nuclear membrane translocation and localization has been

    reported previously [34,35]. Recently, Millers laboratory

    has shown that Mu functions as an NLS, since a fusion

    of Mu with -galactosidase was shown to efficiently

    accumulate in the nucleus [36]. This is also supported

    by our results where all the three fusion proteins have

    been shown to internalize efficiently and localize in the

    nucleus thereby transfecting various cell lines.

    TAT is probably the most investigated among PTDs and

    extensive literature is available on its ability to deliverproteins and genes into cells [3739]. The domain that

    confers this unique property to TAT is the stretch of

    YGRKKRRQRRR, rich in arginine and lysine, responsible

    for membrane transduction and also nuclear localization

    [38]. Mu-Mu alone was more efficient than Tat-Mu

    or Mu in binding with the DNA as seen from the

    DNA-binding assays and EtBr-exclusion experiments. The

    binding ability of Mu is also reflected in its ability to

    bring about transfection. Reporter gene expressions with

    the three peptides indicated that expression levels are

    more or less comparable and correlate with the increased

    uptake as seen from the confocal experiments. Although

    all the three peptides show efficient internalization ofplasmid DNA, these have been demonstrated to be more

    efficient when combined with cationic lipids. Peptides

    when combined with lipids have been shown to be more

    efficient in bringing about reporter gene expression than

    peptides alone.

    For more than a decade, linear poly-l-lysine has

    been used in gene delivery. Modifications or variations

    in the length of the positively charged polymeric

    scaffold can have a varying effect on transfection

    efficiency thereby contributing to the development of

    the oligolysine molecules also as gene delivery agents.

    Several studies have employed poly-l-lysine of differentsizes for binding and condensation. These complexes are

    effective at concentrations which are usually toxic to

    cells [40 42]. Although poly-l-lysine served as a good

    example, subsequent modification was very essential

    to increase its utility in gene delivery. A stretch of

    positive charges alone may not be sufficient for enhancing

    transfection. Also, the presence of nuclear localization

    does not promote transfection without adequate nucleic

    acid charge neutralization and condensation as well.

    In all the reported instances, the inclusion of peptides or

    cationic polymers has resulted in more uniform complexes

    of 100200 nm size compared to much larger particles

    that result from complexes prepared with cationic lipids.

    We have also observed that the size of DPL is 150 nm

    compared to a few hundreds of nanometers in the

    case of DL prepared with DC-Chol : DOPE and detailed

    investigations are underway. Though it is expected

    that the cellular uptake of the DPL is mediated by

    endocytosis, it is not very clear how various components

    of DPL interact with various organelles inside the cell.

    Preliminary transfection data obtained by the addition of

    chloroquine, a lysosomal disrupting agent, enhanced thereporter gene expression significantly (data not reported)

    indicating the involvement of endocytotic pathways in

    the uptake of DPL. These results demonstrate that the

    internalization of the DNA is enhanced in the presence

    of peptide. Although we observed clear enhancements

    in transfection efficiencies with DP complexes, the exact

    contributions of TAT-Mu, Mu and Mu-Mu in enhancing

    DNA delivery needs to be investigated. Nevertheless, the

    observed uptake, co-localization and immunolocalization

    data when combined would explain the efficiency of

    transfection observed by us.

    Cationic peptides, although less commonly used, arehowever gaining attention due to their versatility and

    advantages. By combining cues from viral proteins

    with that of current transfection protocols that employ

    cationic lipids, novelty combined with multifunctionality

    is introduced making them more stable, potential, next-

    generation formulations. Cationic lipids do not offer all

    the features that are required for a unique delivery system

    and most current methods seek to generate formulations

    not confined to cationic lipids alone. The benefit of using

    recombinant peptides is the versatility that can be added

    to the recombinant molecule by genetic engineering

    methods to suit various cell types. The addition of cell-

    specific epitopes would make these heterologous fusions

    therapeutically significant particularly in differentiated

    cells as NLS-like features characterize these constructs.

    This would also advance the development of vectors

    where a combination vector system could be reconstituted

    depending on desired protocol.

    Conclusions

    Recombinant proteins that are heterologous fusions with

    DNA binding and nuclear localization epitopes of viral ori-gin, generated in this study, have considerable potential

    to facilitate DNA delivery and enhance transfection. By

    using this strategy with cues from viral proteins, versatility

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    Recombinant Fusion Proteins Mediate Efficient Gene Delivery 285

    to the molecule can be added to the modules rapidly in a

    combinatorial fashion. Unique cell-targeting moieties can

    be added to these basic constructs thereby making them a

    more specific-potential transfecting agent to suit all gene

    delivery strategies. An important aspect that emerges from

    this study is that although the recombinant proteins have

    been shown to efficiently transfect actively dividing cells,

    the presence of NLS domains in these fusion proteins may

    confer the potential to transfect non-dividing/resting cells.

    This would impact strategies using ex vivo cell therapies.

    In situations where the nuclear membrane dissolution

    does not take place, the complexes could gain entry into

    the nucleus and subsequently become available to the

    transcriptional machinery. This would have tremendous

    therapeutic significance and would advance studies in the

    direction of gene medicine.

    Acknowledgements

    Theauthors would like to thank theDepartment of Biotechnology

    for a DBT fellowship to RR. We would also like to thank

    Prof. David Dean, Northwestern University, Chicago, for going

    through the initial draft of the manuscript. We acknowledge

    Soumya Sudhakar, summer student, in the preparation of the

    pMu construct.

    Supplementary Material

    The supplementary electronic material for this paper

    is available in Wiley InterScience at: http://www.interscience.wiley.com/jpages/1099-498X/suppmat/.

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    Supplementary information for the online versionof the paper Recombinant fusion proteins TAT-Mu,

    Mu and Mu-Mu mediate efficient non-viral genedelivery

    Figure S1.