BACKGROUND
the development of efficient and non-toxic vectors for cell-specific gene delivery is a major challenge in gene therapeutic research.
a significant progress has been made in the construction of non-viral vectors that combine different functions required for gene transfer in an artificial complex, as the potential advantages of such a system include ease of use, cost-effective large-scale manufacture, purity, homogeneity, as well as fewer and more well-defined regulatory issues
<cit> .
however, the alternative approaches relying on the activities of natural or recombinant dna-carrier proteins to achieve uptake and intracellular delivery of plasmid dna has not been developed.
transferrin  plays an important role in the cellular iron uptake.
once tf binds with transferrin receptor  on the cell surface, it is ingested into the endosome under acidic condition.
during this process, iron is released and the tfr-apo-tf complexes then re-circulate into the cell surface.
upon disassociation of tfr, apo-tf regains the ability of binding to iron again.
tfr is a cell membrane-associated glycoprotein involved in the cellular uptake of iron and in the regulation of cell growth
<cit>  and it is preferentially expressed in cells with high potential for proliferation.
therefore, the remarkable and stable tfr expression can be detected in various tumor cells such as hepatoma carcinoma cells and leukemia cells
<cit> .
given its abundance in malignant tissues and its significance in the cellular iron uptake, tfr could therefore be used as a biomarker for tumor cells in addition to its relevance in cancer and its extracellular accessibility.
these characteristics also render tfr to be an excellent antigen for antibody-based cancer therapy
<cit> .
indeed, many tfr-specific antibodies  have been developed and employed to kill the malignant cells in vitro and in vivo <cit> .
previously, we have successfully developed several antibodies against tfr including the human-mouse chimeric antibody, intracellular antibody and bivalent single chain variable fragment  antibody
<cit> .
all of these antibodies displayed tumor-specific biodistribution with substantial antitumor activity.
we also employed a transferrin-polyethylenimine  delivery system to carry hif-1α shrnas into distant tumors.
our studies demonstrated that tfr-mediated endocytosis could induce hif-1α silencing and resulted in impaired xenograft growth of melanoma in vivo <cit> .
together, these studies support the possibility that antibodies specific for tfr could be used as a feasible carrier of genes to target for tumor therapy or diagnosis.
however, in comparison with the whole antibody or transferin molecule, the scfv has a smaller size for a better penetration into tumor cells.
indeed, low molecule weight single-stranded antibody can be conjugated with various therapeutic or diagnostic molecules to generate targeting complex.
also, the internalized tfr-complex can facilitate the development of tumor therapy or imaging.
the nuclear protein gal <dig> is a positive regulator for galactose-induced gene expressions such as gal <dig>  gal <dig>  gal <dig>  gal <dig>  and mel1
<cit> .
the high-affinity of well-characterized of gal <dig> dna-binding has previously been shown to be retained when placed in the context of a heterologous fusion partner or used to enhance gene delivery through conjugation of ligand and other cationic polymers
<cit> .
gal <dig> possesses high binding affinity for a specific 17-bp oligonucleotide sequence  and acts as a nuclear localization signal
<cit> .
most importantly, its specific dna-binding domain  can bind to the plasmid containing an anti-tumor gene or a specific target gene for imaging.
these properties make it a good candidate for use as a vehicle for gene transfer.
in the present study, we developed a particularly appealing approach for the delivery of genes through the use of recombinant protein-based vehicles which consists of a dna-binding motif fused to a cell-binding tfr-scfv.
the tfrscfv-gal <dig> protein has a dual function: specific dna binding via gal4-dbd and the gal4rec of the dna plasmid, and intracellular delivery of the target dna by tfr-scfv transport.
this tfrscfv-gal <dig> fusion-mediated dna delivery system effectively transduced the pgfp-plasmid and induced gfp expression in mammalian cells.
we further explored its feasibility for application of tumor-targeted drug delivery and in vivo imaging.
RESULTS
construction of tfrscfv-gal4pet expression vector, expression, purification and renaturation of tfrscfv-gal <dig> fusion protein in e. coli
the plasmid for tfrscfv-gal <dig> fusion protein was constructed as described.
sequencing analysis of the inserts matched exactly with the targeted gene sequences in the database .
the full-length sequence for the tfrscfv-gal <dig> coding region was identical with the ncoi-scfv-ecori-gal4-noti sequence .
high levels tfrscfv-gal <dig> of expression of was induced in pet28/tfrscfv-gal <dig> transformed bl <dig>  e. coli upon the addition of 1mm iptg.
the optimal induction temperature of 30°c was determined to yield the maximum level of protein expression as shown in lane  <dig> of figure
2a.
the majority of the fusion protein  was found not to be soluble but to be contained within the inclusion bodies.
therefore, the tfrscfv-gal <dig> containing the his-tagged was purified from the inclusion bodies as described in “methods.” as shown in figure
2b, the tfrscfv-gal <dig> fusion protein was then purified from cell lysates under denaturing conditions using the ni–nta column and re-natured by urea gradient dialysis.
sds-page showed a single band of the re-folded and purified fusion protein with an approximate molecular weight of  <dig> ~ <dig>  kda, in agreement with the expected molecular weight  for the recombinant fusion protein, as well as the highest expression were noted 6h after iptg induction at 30°c.
in contrast, no reactive band was detected in bl <dig> e. coli transformed with an empty vector .
the concentration of re-natured protein was  <dig>  mg/ml as measured by the bca protein assay.
immunoreactivity of tfrscfv-gal <dig> with various tumor cell lines
the binding of tfrscfv-gal <dig> to the tfr was studied by flow cytometry using the tfr overexpression on tumor cell lines.
we next sought to determine the antigen-binding activity for the tfrscfv-gal <dig> fusion protein with various tumor cells.
flow cytometry analysis was employed for this purpose.
it was noted that only the tfrscfv-gal <dig> group  and the positive controls  showed positive results by flow cytometry assay, while the negative controls failed to detect any positive cells .
as shown in figure
3b, the binding rate for tfrscfv-gal <dig> fusion protein with  <dig> different tumor cells varied between  <dig> % to  <dig> %, and there was a significant difference in terms of the binding rate between the tfrscfv-gal <dig> group and the negative control group .
it showed that the purified tfrscfv-gal <dig> retained its immunoreactivity comparable with that of the parental anti-tfr mab.
ag-binding potency of tfrscfv-gal <dig> with various tissue microarray  sections
according to the standard of tma sections described in “methods”, for gastric cancer tma sections , the available cases was  <dig> for tfrscfv-gal <dig> fusion protein group,  <dig> for mouse anti-tfr antibody group, were  <dig> and  <dig> in mouse anti-gal <dig> antibody group and mouse nonspecific igg group, respectively.
the available ratios were 85%, 70%, 85% and 83%, respectively.
for gastric cancer tma sections, the positive ratios were  <dig> %,  <dig> %, 0% and 0%, respectively .
for two breast cancer tma sections , the available cases were  <dig>   <dig>   <dig> and  <dig>  respectively; the available ratios were 98%, 96%, 97% and 96%, respectively.
for two breast cancer tma sections, the positive ratios were  <dig> %,  <dig> %, 0% and 0%, respectively .
the available ratios and positive ratios for tma sections were showed in table
<dig>  there was a significant difference for the positive ratio between the tfrscfv-gal <dig> group and the negative control group  .
<dig> showed the statistic data of all tissue in immunohistology results.
in contrast to the normal tissue sections, only  <dig> liver cases for the normal tissue sections showed weakly positive for the tfrscfv-gal <dig> fusion protein group and mouse anti-tfr antibody group, while other tissues including the heart, spleen, adrenal cortex, blood vessels and stomach were negative for both groups .
of note, all normal tissue sections were negative for the mouse anti-gal <dig> group and mouse nonspecific igg group.
reactivity of tfrscfv-gal <dig> with anti-gal <dig> antibody
to measure the binding activity of gal <dig> domain for the purified tfrscfv-gal <dig> fusion protein, we performed the elisa analysis.
the results revealed that both tfrscfv-gal <dig> fusion protein and gal <dig> protein bound to a rabbit anti-gal <dig> antibody coated on the surface of a microtiter plate in a dose-dependent manner.
furthermore, the od <dig> value increased with the parallel increase of the protein concentration  as shown in figure
<dig>  thus, the gal <dig> portion among tfrscfv-gal <dig> fusion protein retained its high avidity for binding to rabbit anti-gal <dig> antibody and it suggested the fusion protein re-naturation did not impair the gla <dig> activity.
functional characterization of the fusion proteins
the ability of protein-dna binding was used to assess the biological activity of gla <dig> in tfrscfv-gal <dig> fusion protein.
in the presence of the proper conjugating condition, the purified protein were then added to the plasmids with varying concentration to form the protein-dna complex which were analyzed by electrophoresis on  <dig> % gradient natural  page followed by western analysis, it showed that only one protein band could be seen but the moved protein band was dose-dependent with addition of dna plasmids with the optimal molar ratio of protein to plasmid was 1: <dig>  .
these constitutive proteins also served as a basis of comparison for the activities of the fusion protein.
flow cytometry was used to analyze the transfection efficiency of the protein-dna complex mediated by tfrscfv portion.
after a 48-hour incubation, expression of the gfp reporter gene was found in % of hepg- <dig> cells that had been transfected with the mammalian expression plasmid of gal4rec-gfp-pges in the complex group , in % and % of cells transfected with control and in % transfected with naked dna plasmid.
it indicated that the endocytosis of the tfrscfv-targeted complex to tumor cells was significantly higher than those without tfrscfv as target molecule.
in addition, figure
<dig> in columns b,d,f, and g also illustrated that the capability of tfrscfv-gal <dig> to mediate transfer gal4rec-gfp-pges expression plasmid into hepg <dig> cells as compared to gfp-pges plasmid without gla <dig>  tfrscfv alone did not exhibit any dna-binding capacity, confirming that the dna-binding capacity of tfrscfv-gal <dig> was strictly a function of the gal <dig> component.
discussion
recently, non-viral vectors are under intense investigation as a safer alternative for gene therapy.
for successful delivery, efficient and specific targeting of an active agent to the desired site is a critical factor for overcoming many barriers to carry dna to nuclear localization and transcription
<cit> .
some of the most common non-viral vectors include polyethylenimine, dendrimers, chitosan, polylysine, and many types of peptides, which are generally cationic in nature and able to interact with plasmid dna through electrostatic interactions
<cit> .
peptide-based vectors are advantageous over other non-viral strategies in that attachment of a peptide ligand to the polyplex will allow targeting to specific receptors and/or specific cell types, particularly in cancer cells
<cit> .
however, an important drawback of the chemical coupling procedure is the difficulty in producing a reproducible and homogeneous product.
genetic engineering provides an alternative approach for large scale production of homogeneous ab-gla <dig> fusion proteins.
previous studies have consistently suggested that tfr is expressed more abundantly in malignant tissues than that in their healthy counterparts
<cit> .
similarly, serum levels for soluble transferrin receptor  in patients with hepatoma and haematologic malignancies are significantly increased as compared with those of normal controls
<cit> .
administration of anti-tfr antibody suppressed tumor cells growth in vitro <cit> .
most importantly, tfr served as an endocytotic receptor which could carry the vector for uptake by the tumor cells
<cit> .
based on these findings, anti-tfr antibody is now considered as an alternative for diagnosis by imaging localization and for treatment targeting tumors
<cit> .
the tfr-specific monoclonal or chimeric antibodies  have been developed and used as an alternative therapeutic approach to kill malignant cells both in vitro and in vivo <cit> .
although promising, this approach requires that unique chimeric molecules be constructed for each specific application or incapability to penetrate tissues for the high molecule weight of complete human-mouse chimeric tfr antibody.
thus, it also faces a formidable challenge which sometimes can lead to a decreased activity or loss of activity of one or both of the covalently conjugated partners.
to overcome these limitations, it is therefore desirable to develop a universal delivery system that eliminates the need for a specific construct for each individual application.
the single chain antibody possesses the property to be integrated with different proteins for preparing some tumor targeting vectors .
another critical feature for the single chain antibody is the low molecular weight , which renders it with enhanced capability to penetrate tissues and cells.
if tfrscfv is inserted into a eukaryocyte expression vector, the vector would be much more efficiently intaken by malignant cells.
tfrscfv can be expressed in tumor cells by the vector.
by using this approach, the efficiency for cancer therapy has been significantly improved and the dosage for anti-tumor drugs can also be reduced
<cit> .
gal <dig> is an 881-amino-acid protein with a zn-cys binuclear cluster type dna-binding domain which is a positive regulator for galactose-induced gene expression
<cit> .
the dna-binding domain can specifically bind to a 17bp recognizing sequence motif .
previous studies demonstrated that the fusion protein gal4/inv could be used in a dna delivery system to target tumor cells
<cit> .
therefore, gal <dig> could be used as a carrier for a plasmid containing the gal4rec gene sequence.
in the current investigation, we generated and characterized bifunctionally active a tfrscfv-gal <dig> fusion derived from plasmid puc <dig> and pabgal <dig>  tfrscfv-gal <dig> with hexahistidine residues at the n-terminus were expressed in e. coli and recovered from the inclusion bodies with subsequent application of metal-chelate affinity chromatography.
the fusion protein described in the present study is the first example of recombinant tfr single-chain monovalent antibodies with gal <dig> dna-binding activity produced in prokaryotic expression system, whereas either constituent component of recombinant protein showed individual activity through the following assays.
fcm assay revealed that the tfrscfv-gal <dig> fusion protein is capable of binding to tumor cells, whose immune-reactivity is consistent with the parental anti-tfr monoclonal antibody.
therefore, our studies provided direct evidence that tfrscfv -gal <dig> fusion protein, instead of gal <dig> protein, could bind to various tumor cells.
we also found that the binding rate for the tfrscfv-gal <dig> fusion protein with seven different tumor cell lines varied between  <dig> % and  <dig> %.
the differences for the binding rate may account for tfr being found expressed preferentially in the proliferation or differentiation stage of these cells rather than g <dig> stage, which was consistent with the results from crepin r et al.
<cit>  and our own results previously
<cit> .
elisa analysis indicated that the tfrscfv-gal <dig> fusion protein can bind to anti-gal <dig> antibody.
therefore, it showed that the fusion protein retained gla <dig> activity and could also be served as a marker to evaluate the binding capability of tfrscfv with tumor cells.
by using a mouse antibody against gal <dig> and a fitc-labeled anti-mouse antibody, fcm assay was applied to detect the binding capability of the fusion protein with cells expressing tfr.
similarly, an hrp-labeled anti-mouse antibody could be used for detecting the binding capability of the fusion protein with tissues through tma assay.
immunohistochemical studies in tma indicated that both tfrscfv-gal <dig> fusion protein and the mouse anti-tfr monoclonal antibody could bind to gastric cancer cells as well as breast cancer cells.
in sharp contrast, neither gal <dig> protein nor mouse non-specific igg showed such binding activity, indicating that tfrscfv-gal <dig> fusion protein binding to human gastric cancer cells and human breast cancer cells is through the tfrscfv domain but not gal <dig> protein domain.
furthermore, except for  <dig> normal liver tissues with weekly binding activity, tfrscfv-gal <dig> fusion protein and the mouse anti-tfr antibody failed to show any binding activity to normal tissues, suggesting that tfrscfv-gal <dig> fusion protein was relatively specific to tumor tissues.
we designed the dna-binding functional assay to further determine the fusion protein function.
after conjugating the tfrscfv-gal <dig> to the plasmid gal4rec-pges which was engineered to include a specific long sequence in the upstream activating sequence  and reporter gene gfp, protein-dna complex incubated with hepatic carcinoma cells hepg <dig>  and the gfp expression in cells was detected by fluorescence microscopy.
cells treated with complex tfrscfv-gal4-gal4rec-gfp-pges showed a strong fluorescence signal, but cells treated with gal <dig> lacking the tfrscfv, or those treated with tfrscfv without gla <dig> portion, or naked dna alone almost did not fluoresce.
these data demonstrated that this process was in fact gla4-dna recognition and then mediated through the attachment of the scfv to the transferrin receptor on the tumor cells, and the fusion protein has the unique capacity to mediate gene transfer.
taken together, this protein-dna may be considered as a promising candidate for developing novel tumor-targeted gene delivery for systemic gene therapy of various human cancers.
CONCLUSIONS
we generated a novel dna delivery vehicle containing single chain variable fragment of anti-transferrin receptor  and gla dna binding domain .
gal4-dbd is capable of binding dna containing its specific binding sequence, and tfrscfv binding tfr on tumor cells is able to uptake by tumor cells through cell membranes to deliver dna.
this approach entailed covalently conjugating the fusion protein tfrscfv-gal <dig> to the plasmid via a gal <dig> at the 3’-end of the protein recognizing gal4rec on the transfected plasmid .
our results showed that this recombinant protein does not impair the immunological activity or targeting ability of the tfrscfv, as well as the functional avidity of gla <dig> recognizing the gal4rec sequence.
the tfrscfv-gal <dig> targeted the protein-dna complex to tumor cells and enhanced the transfection efficiencies in vitro, but it did not exhibit the binding activity for normal tissues.
the potential utility and appeal of this peptide-guided gene delivery lies with its internalization, the ability to control the nature of its constituent parts, and ease of generation.
briefly, this investigation provided basis for tfrscfv-gal <dig> applications of orientation, tracing, imaging and targeted therapy on tumors.

 the development of efficient and non-toxic vectors for cell-specific gene delivery is a major challenge in gene therapeutic research. a significant progress has been made in the construction of non-viral vectors that combine different functions required for gene transfer in an artificial complex, as the potential advantages of such a system include ease of use, cost-effective large-scale manufacture, purity, homogeneity, as well as fewer and more well-defined regulatory issues. however, the alternative approaches relying on the activities of natural or recombinant dna-carrier proteins to achieve uptake and intracellular delivery of plasmid dna has not been developed. transferrin plays an important role in the cellular iron uptake. once tf binds with transferrin receptor on the cell surface, it is ingested into the endosome under acidic condition. during this process, iron is released and the tfr-apo-tf complexes then re-circulate into the cell surface. upon disassociation of tfr, apo-tf regains the ability of binding to iron again. tfr is a cell membrane-associated glycoprotein involved in the cellular uptake of iron and in the regulation of cell growth and it is preferentially expressed in cells with high potential for proliferation. therefore, the remarkable and stable tfr expression can be detected in various tumor cells such as hepatoma carcinoma cells and leukemia cells. given its abundance in malignant tissues and its significance in the cellular iron uptake, tfr could therefore be used as a biomarker for tumor cells in addition to its relevance in cancer and its extracellular accessibility. these characteristics also render tfr to be an excellent antigen for antibody-based cancer therapy. indeed, many tfr-specific antibodies have been developed and employed to kill the malignant cells in vitro and in vivo. previously, we have successfully developed several antibodies against tfr including the human-mouse chimeric antibody, intracellular antibody and bivalent single chain variable fragment antibody. all of these antibodies displayed tumor-specific biodistribution with substantial antitumor activity. we also employed a transferrin-polyethylenimine delivery system to carry hif-1α shrnas into distant tumors. our studies demonstrated that tfr-mediated endocytosis could induce hif-1α silencing and resulted in impaired xenograft growth of melanoma in vivo. together, these studies support the possibility that antibodies specific for tfr could be used as a feasible carrier of genes to target for tumor therapy or diagnosis. however, in comparison with the whole antibody or transferin molecule, the scfv has a smaller size for a better penetration into tumor cells. indeed, low molecule weight single-stranded antibody can be conjugated with various therapeutic or diagnostic molecules to generate targeting complex. also, the internalized tfr-complex can facilitate the development of tumor therapy or imaging. the nuclear protein gal is a positive regulator for galactose-induced gene expressions such as gal gal gal gal and mel1. the high-affinity of well-characterized of gal dna-binding has previously been shown to be retained when placed in the context of a heterologous fusion partner or used to enhance gene delivery through conjugation of ligand and other cationic polymers. gal possesses high binding affinity for a specific 17-bp oligonucleotide sequence and acts as a nuclear localization signal. most importantly, its specific dna-binding domain can bind to the plasmid containing an anti-tumor gene or a specific target gene for imaging. these properties make it a good candidate for use as a vehicle for gene transfer. in the present study, we developed a particularly appealing approach for the delivery of genes through the use of recombinant protein-based vehicles which consists of a dna-binding motif fused to a cell-binding tfr-scfv. the tfrscfv-gal protein has a dual function: specific dna binding via gal4-dbd and the gal4rec of the dna plasmid, and intracellular delivery of the target dna by tfr-scfv transport. this tfrscfv-gal fusion-mediated dna delivery system effectively transduced the pgfp-plasmid and induced gfp expression in mammalian cells. we further explored its feasibility for application of tumor-targeted drug delivery and in vivo imaging.  construction of tfrscfv-gal4pet expression vector, expression, purification and renaturation of tfrscfv-gal fusion protein in e. coli the plasmid for tfrscfv-gal fusion protein was constructed as described. sequencing analysis of the inserts matched exactly with the targeted gene sequences in the database. the full-length sequence for the tfrscfv-gal coding region was identical with the ncoi-scfv-ecori-gal4-noti sequence. high levels tfrscfv-gal of expression of was induced in pet28/tfrscfv-gal transformed bl e. coli upon the addition of 1mm iptg. the optimal induction temperature of 30°c was determined to yield the maximum level of protein expression as shown in lane of figure 2a. the majority of the fusion protein was found not to be soluble but to be contained within the inclusion bodies. therefore, the tfrscfv-gal containing the his-tagged was purified from the inclusion bodies as described in “methods.” as shown in figure 2b, the tfrscfv-gal fusion protein was then purified from cell lysates under denaturing conditions using the ni–nta column and re-natured by urea gradient dialysis. sds-page showed a single band of the re-folded and purified fusion protein with an approximate molecular weight of ~ kda, in agreement with the expected molecular weight for the recombinant fusion protein, as well as the highest expression were noted 6h after iptg induction at 30°c. in contrast, no reactive band was detected in bl e. coli transformed with an empty vector. the concentration of re-natured protein was mg/ml as measured by the bca protein assay. immunoreactivity of tfrscfv-gal with various tumor cell lines the binding of tfrscfv-gal to the tfr was studied by flow cytometry using the tfr overexpression on tumor cell lines. we next sought to determine the antigen-binding activity for the tfrscfv-gal fusion protein with various tumor cells. flow cytometry analysis was employed for this purpose. it was noted that only the tfrscfv-gal group and the positive controls showed positive results by flow cytometry assay, while the negative controls failed to detect any positive cells. as shown in figure 3b, the binding rate for tfrscfv-gal fusion protein with different tumor cells varied between % to %, and there was a significant difference in terms of the binding rate between the tfrscfv-gal group and the negative control group. it showed that the purified tfrscfv-gal retained its immunoreactivity comparable with that of the parental anti-tfr mab. ag-binding potency of tfrscfv-gal with various tissue microarray sections according to the standard of tma sections described in “methods”, for gastric cancer tma sections, the available cases was for tfrscfv-gal fusion protein group, for mouse anti-tfr antibody group, were and in mouse anti-gal antibody group and mouse nonspecific igg group, respectively. the available ratios were 85%, 70%, 85% and 83%, respectively. for gastric cancer tma sections, the positive ratios were %, %, 0% and 0%, respectively. for two breast cancer tma sections, the available cases were and respectively; the available ratios were 98%, 96%, 97% and 96%, respectively. for two breast cancer tma sections, the positive ratios were %, %, 0% and 0%, respectively. the available ratios and positive ratios for tma sections were showed in table there was a significant difference for the positive ratio between the tfrscfv-gal group and the negative control group. showed the statistic data of all tissue in immunohistology results. in contrast to the normal tissue sections, only liver cases for the normal tissue sections showed weakly positive for the tfrscfv-gal fusion protein group and mouse anti-tfr antibody group, while other tissues including the heart, spleen, adrenal cortex, blood vessels and stomach were negative for both groups. of note, all normal tissue sections were negative for the mouse anti-gal group and mouse nonspecific igg group. reactivity of tfrscfv-gal with anti-gal antibody to measure the binding activity of gal domain for the purified tfrscfv-gal fusion protein, we performed the elisa analysis. the results revealed that both tfrscfv-gal fusion protein and gal protein bound to a rabbit anti-gal antibody coated on the surface of a microtiter plate in a dose-dependent manner. furthermore, the od value increased with the parallel increase of the protein concentration as shown in figure thus, the gal portion among tfrscfv-gal fusion protein retained its high avidity for binding to rabbit anti-gal antibody and it suggested the fusion protein re-naturation did not impair the gla activity. functional characterization of the fusion proteins the ability of protein-dna binding was used to assess the biological activity of gla in tfrscfv-gal fusion protein. in the presence of the proper conjugating condition, the purified protein were then added to the plasmids with varying concentration to form the protein-dna complex which were analyzed by electrophoresis on % gradient natural page followed by western analysis, it showed that only one protein band could be seen but the moved protein band was dose-dependent with addition of dna plasmids with the optimal molar ratio of protein to plasmid was 1:. these constitutive proteins also served as a basis of comparison for the activities of the fusion protein. flow cytometry was used to analyze the transfection efficiency of the protein-dna complex mediated by tfrscfv portion. after a 48-hour incubation, expression of the gfp reporter gene was found in % of hepg- cells that had been transfected with the mammalian expression plasmid of gal4rec-gfp-pges in the complex group, in % and % of cells transfected with control and in % transfected with naked dna plasmid. it indicated that the endocytosis of the tfrscfv-targeted complex to tumor cells was significantly higher than those without tfrscfv as target molecule. in addition, figure in columns b,d,f, and g also illustrated that the capability of tfrscfv-gal to mediate transfer gal4rec-gfp-pges expression plasmid into hepg cells as compared to gfp-pges plasmid without gla tfrscfv alone did not exhibit any dna-binding capacity, confirming that the dna-binding capacity of tfrscfv-gal was strictly a function of the gal component. discussion recently, non-viral vectors are under intense investigation as a safer alternative for gene therapy. for successful delivery, efficient and specific targeting of an active agent to the desired site is a critical factor for overcoming many barriers to carry dna to nuclear localization and transcription. some of the most common non-viral vectors include polyethylenimine, dendrimers, chitosan, polylysine, and many types of peptides, which are generally cationic in nature and able to interact with plasmid dna through electrostatic interactions. peptide-based vectors are advantageous over other non-viral strategies in that attachment of a peptide ligand to the polyplex will allow targeting to specific receptors and/or specific cell types, particularly in cancer cells. however, an important drawback of the chemical coupling procedure is the difficulty in producing a reproducible and homogeneous product. genetic engineering provides an alternative approach for large scale production of homogeneous ab-gla fusion proteins. previous studies have consistently suggested that tfr is expressed more abundantly in malignant tissues than that in their healthy counterparts. similarly, serum levels for soluble transferrin receptor in patients with hepatoma and haematologic malignancies are significantly increased as compared with those of normal controls. administration of anti-tfr antibody suppressed tumor cells growth in vitro. most importantly, tfr served as an endocytotic receptor which could carry the vector for uptake by the tumor cells. based on these findings, anti-tfr antibody is now considered as an alternative for diagnosis by imaging localization and for treatment targeting tumors. the tfr-specific monoclonal or chimeric antibodies have been developed and used as an alternative therapeutic approach to kill malignant cells both in vitro and in vivo. although promising, this approach requires that unique chimeric molecules be constructed for each specific application or incapability to penetrate tissues for the high molecule weight of complete human-mouse chimeric tfr antibody. thus, it also faces a formidable challenge which sometimes can lead to a decreased activity or loss of activity of one or both of the covalently conjugated partners. to overcome these limitations, it is therefore desirable to develop a universal delivery system that eliminates the need for a specific construct for each individual application. the single chain antibody possesses the property to be integrated with different proteins for preparing some tumor targeting vectors. another critical feature for the single chain antibody is the low molecular weight, which renders it with enhanced capability to penetrate tissues and cells. if tfrscfv is inserted into a eukaryocyte expression vector, the vector would be much more efficiently intaken by malignant cells. tfrscfv can be expressed in tumor cells by the vector. by using this approach, the efficiency for cancer therapy has been significantly improved and the dosage for anti-tumor drugs can also be reduced. gal is an 881-amino-acid protein with a zn-cys binuclear cluster type dna-binding domain which is a positive regulator for galactose-induced gene expression. the dna-binding domain can specifically bind to a 17bp recognizing sequence motif. previous studies demonstrated that the fusion protein gal4/inv could be used in a dna delivery system to target tumor cells. therefore, gal could be used as a carrier for a plasmid containing the gal4rec gene sequence. in the current investigation, we generated and characterized bifunctionally active a tfrscfv-gal fusion derived from plasmid puc and pabgal tfrscfv-gal with hexahistidine residues at the n-terminus were expressed in e. coli and recovered from the inclusion bodies with subsequent application of metal-chelate affinity chromatography. the fusion protein described in the present study is the first example of recombinant tfr single-chain monovalent antibodies with gal dna-binding activity produced in prokaryotic expression system, whereas either constituent component of recombinant protein showed individual activity through the following assays. fcm assay revealed that the tfrscfv-gal fusion protein is capable of binding to tumor cells, whose immune-reactivity is consistent with the parental anti-tfr monoclonal antibody. therefore, our studies provided direct evidence that tfrscfv -gal fusion protein, instead of gal protein, could bind to various tumor cells. we also found that the binding rate for the tfrscfv-gal fusion protein with seven different tumor cell lines varied between % and %. the differences for the binding rate may account for tfr being found expressed preferentially in the proliferation or differentiation stage of these cells rather than g stage, which was consistent with the results from crepin r et al. and our own results previously. elisa analysis indicated that the tfrscfv-gal fusion protein can bind to anti-gal antibody. therefore, it showed that the fusion protein retained gla activity and could also be served as a marker to evaluate the binding capability of tfrscfv with tumor cells. by using a mouse antibody against gal and a fitc-labeled anti-mouse antibody, fcm assay was applied to detect the binding capability of the fusion protein with cells expressing tfr. similarly, an hrp-labeled anti-mouse antibody could be used for detecting the binding capability of the fusion protein with tissues through tma assay. immunohistochemical studies in tma indicated that both tfrscfv-gal fusion protein and the mouse anti-tfr monoclonal antibody could bind to gastric cancer cells as well as breast cancer cells. in sharp contrast, neither gal protein nor mouse non-specific igg showed such binding activity, indicating that tfrscfv-gal fusion protein binding to human gastric cancer cells and human breast cancer cells is through the tfrscfv domain but not gal protein domain. furthermore, except for normal liver tissues with weekly binding activity, tfrscfv-gal fusion protein and the mouse anti-tfr antibody failed to show any binding activity to normal tissues, suggesting that tfrscfv-gal fusion protein was relatively specific to tumor tissues. we designed the dna-binding functional assay to further determine the fusion protein function. after conjugating the tfrscfv-gal to the plasmid gal4rec-pges which was engineered to include a specific long sequence in the upstream activating sequence and reporter gene gfp, protein-dna complex incubated with hepatic carcinoma cells hepg and the gfp expression in cells was detected by fluorescence microscopy. cells treated with complex tfrscfv-gal4-gal4rec-gfp-pges showed a strong fluorescence signal, but cells treated with gal lacking the tfrscfv, or those treated with tfrscfv without gla portion, or naked dna alone almost did not fluoresce. these data demonstrated that this process was in fact gla4-dna recognition and then mediated through the attachment of the scfv to the transferrin receptor on the tumor cells, and the fusion protein has the unique capacity to mediate gene transfer. taken together, this protein-dna may be considered as a promising candidate for developing novel tumor-targeted gene delivery for systemic gene therapy of various human cancers.  we generated a novel dna delivery vehicle containing single chain variable fragment of anti-transferrin receptor and gla dna binding domain. gal4-dbd is capable of binding dna containing its specific binding sequence, and tfrscfv binding tfr on tumor cells is able to uptake by tumor cells through cell membranes to deliver dna. this approach entailed covalently conjugating the fusion protein tfrscfv-gal to the plasmid via a gal at the 3’-end of the protein recognizing gal4rec on the transfected plasmid. our results showed that this recombinant protein does not impair the immunological activity or targeting ability of the tfrscfv, as well as the functional avidity of gla recognizing the gal4rec sequence. the tfrscfv-gal targeted the protein-dna complex to tumor cells and enhanced the transfection efficiencies in vitro, but it did not exhibit the binding activity for normal tissues. the potential utility and appeal of this peptide-guided gene delivery lies with its internalization, the ability to control the nature of its constituent parts, and ease of generation. briefly, this investigation provided basis for tfrscfv-gal applications of orientation, tracing, imaging and targeted therapy on tumors. 
the gal4-dna functional assay through the gal complementary conjugation with the gal4rec-gfp-pges plasmid to verify the gla activity and gal4rec-recognized specificity functions. the development of vectors for cell-specific gene delivery is a major goal of gene therapeutic strategies. transferrin receptor is an endocytic receptor and identified as tumor relative specific due to its overexpression on most tumor cells or tissues, and tfr binds and intakes of transferrin-iron complex. we have previously generated an anti-tfr single-chain variable fragments of immunoglobulin which were cloned from hybridoma cell line producing antibody against tfr linked with a aa-long linker sequence in the present study, the anti-tfr single-chain antibody was fused to dna-binding domain of the yeast transcription factor gal the recombinant fusion protein, designated as tfrscfv-gal is expected to mediate the entry of dna-protein complex into targeted tumor cells. in biological assays, the antigen-binding activity of the re-natured protein, tfrscfv-gal was confirmed by specific binding to different cancer cells and tumor tissues. the positive binding rates to tumor tissues by tissue microarrays assays were % and %, but it showed weakly binding with hepatic tissue in cases, and normal tissues such as heart, spleen, adrenal cortex blood vessel and stomach. results of our study demonstrated that the biofunctianality of genetically engineered fusion protein, tfrscfv-gal was retained, as the fusion protein could both carry the plasmid of gal4rec-pges and bind tfr on tumour cells. fusion protein tfrscfv-gal was expressed in an e. coli bacterial expression system and was recovered from inclusion bodies with subsequent purification by metal-chelate chromatography. in addition, the re-natured fusion protein tfrscfv-gal was used in an elisa with rabbit anti-gal antibody.
 the development of vectors for cell-specific gene delivery is a major goal of gene therapeutic strategies. transferrin receptor is an endocytic receptor and identified as tumor relative specific due to its overexpression on most tumor cells or tissues, and tfr binds and intakes of transferrin-iron complex. we have previously generated an anti-tfr single-chain variable fragments of immunoglobulin which were cloned from hybridoma cell line producing antibody against tfr linked with a aa-long linker sequence in the present study, the anti-tfr single-chain antibody was fused to dna-binding domain of the yeast transcription factor gal the recombinant fusion protein, designated as tfrscfv-gal is expected to mediate the entry of dna-protein complex into targeted tumor cells.  fusion protein tfrscfv-gal was expressed in an e. coli bacterial expression system and was recovered from inclusion bodies with subsequent purification by metal-chelate chromatography. the resulting proteins were predominantly monomeric and, upon refolding, became a soluble biologically active bifunctional protein. in biological assays, the antigen-binding activity of the re-natured protein, tfrscfv-gal was confirmed by specific binding to different cancer cells and tumor tissues. the cell binding rates, as indicated by flow cytometry analysis, ranged from % to % in seven different human carcinoma cell lines. it showed similar affinity and binding potency as those of parent full-length mouse anti-tfr antibody. the positive binding rates to tumor tissues by tissue microarrays assays were % and %, but it showed weakly binding with hepatic tissue in cases, and normal tissues such as heart, spleen, adrenal cortex blood vessel and stomach. in addition, the re-natured fusion protein tfrscfv-gal was used in an elisa with rabbit anti-gal antibody. the gal4-dna functional assay through the gal complementary conjugation with the gal4rec-gfp-pges plasmid to verify the gla activity and gal4rec-recognized specificity functions. it also shows the complex, tfrscfv-gal4-gal4rec-gfp-pges, could be taken into endochylema to express the green fluorescent protein with to 10-fold transfection efficiency.  results of our study demonstrated that the biofunctianality of genetically engineered fusion protein, tfrscfv-gal was retained, as the fusion protein could both carry the plasmid of gal4rec-pges and bind tfr on tumour cells. this product was able to transfect target cells effectively in an immuno-specific manner, resulting in transient gene expression. this protein that can be applied as an effective therapeutic and diagnostic delivery to the tumor using endogenous membrane transport system with potential widespread utility. 