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. Author manuscript; available in PMC: 2011 Feb 1.
Published in final edited form as: Cancer Gene Ther. 2009 Aug 28;17(2):131–140. doi: 10.1038/cgt.2009.54

Enhancement of Reporter Gene Detection Sensitivity by Insertion of Specific Mini-Peptide-Coding Sequences

Jeffry Cutrera 1, Denada Dibra 1, Xueqing Xia 1, Shulin Li 1,*
PMCID: PMC2808434  NIHMSID: NIHMS127033  PMID: 19713998

Abstract

Two important aspects for gene therapy are to increase the level of gene expression and track the gene delivery site and expression, and a sensitive reporter gene may be one of the options for preclinical studies and possibly for human clinical trials. We report the novel concept of increasing the activity of the gene products. With the insertion of the mini-peptide-coding sequence CWDDWLC into the plasmid DNA of a SEAP reporter gene, we observed vast increases in the enzyme activity in vitro in all murine and human cell lines used. Also, in vivo injection of this CWDDWLC-SEAP encoding gene resulted in the same increases in reporter gene activity, but these increases did not correspond to alterations in the level of the gene products in the serum. Minor sequence changes in this mini-peptide negate the activity increase of the reporter gene. We report the novel concept of increasing the activity of gene products as another method to improve the reporting sensitivity of reporter genes. This improved reporter gene could complement any improved vector for maximizing the reporter sensitivity. Also, this strategy has the potential to be used to discover peptides that improve the activity of therapeutic genes.

Keywords: Reporter gene, SEAP, peptide, vector, in vivo

Introduction

Gene therapy is on the forefront of many areas of biomedical research and therapeutics. The use of gene therapy ranges from the utilization of reporter genes for noninvasive monitoring and gene distribution1–3 to the application of therapeutic genes to treat several diseases such as cancer4–7 and blood disorders8, 9. Advancements in the development of both viral and non-viral delivery systems for gene therapy result in continuous improvements in the efficacy of gene therapy, but several barriers remain which are blocking gene therapy from becoming widely successful.

Secreted alkaline phosphatase (SEAP) is a widely used reporter gene which is known for its resistance to high temperature and L-homoarginine, unlike endogenous alkaline phosphatases. SEAP is used for a variety of in vitro and in vivo assays including interferon activity10, efficacy of antidepressants11, and endoplasmic reticulum stress12, 13. With current strategies, the low level of gene expression of SEAP as well as other reporter genes affects the use of reporter genes and limits their uses to in vitro and small animal models1–3, 12, 14. Similarly, therapeutic applications of gene therapy are hindered by having trouble acquiring therapeutically significant levels of the gene products15, 16. Although improvements in gene delivery and vector design can increase the total level of gene expression, these advancements do not improve the sensitivity per molecule of the gene product.

To increase the level of gene expression, investigators have focused on increasing gene delivery. For instance, several methods to increase the delivery of genes with viral vectors are currently used. The addition of Poly(ethylene glycol) (PEG) motifs to the capsid fibers of viral vectors, a process termed PEGylation, can increase the viral half life by protecting the viral particles from enzymatic degradation and increasing the circulation time. Also, PEG can protect the viral vectors from cell- and humoral-mediated immune responses. By improving the in vivo viral properties, PEG can increase the level of gene delivery by the viral vectors15. Also, modifying the tropism of the viruses by the addition of tissue-targeted ligands can increase the viral load at specific sites which increases the delivery of genes at the desired location15, 17, 18. Similarly, plasmid delivery can be enhanced by delivery via gene gun, electroporation, ultrasound, and many other techniques16.

Besides gene delivery, the level and duration of gene expression may be one of the main limiting factors for successful gene therapy. One of the most important elements that can affect both of these factors is the promoter. The most commonly used promoter was developed from the immediate early promoter of human cytomegalovirus (CMV) because it can increase the level of expression in a wide range of cell types; however, the expression generated from the CMV promoter consistently decreases beginning one to two days after transfection19. Other promoters can increase the level of expression based on the tissue that is transfected. For instance, the muscle creatine promoter can increase expression of SEAP in muscle tissues as well as inhibiting both cell- and humoral- mediated immune responses20. To create more sustained expression, synthetic promoters have been developed by combining promoters and enhancer elements from different sources. One such promoter is the CAGG promoter which consists of the CMV enhancer, β-actin promoter, and a chicken β-actin/rabbit β-actin globin composite intron8. Composite promoters can be developed by using high throughput methods using random assembly of promoter elements to create improved promoters21, 22. Other methods that are used to increase the level and duration of gene expression include reducing the amount of unmethylated CpG dinucleotides6, 23, using codon optimization to remove rarely used codons from plasmid DNA24, 25, and decreasing the size of the vectors by using DNA fragments26. These approaches are all valuable, but current strategies have not overcome all the hurdles to gene therapy that are caused by the low level of gene expression.

To complement these conventional approaches and increase the detection sensitivity of each reporter molecule, here we report a novel concept—increasing the activity of the gene product. Insertion of mini-peptide-coding sequences prior to the stop codon in SEAP-coding DNA drastically alters the enzyme activity of the resulting SEAP enzymes. This effect occurs in all cell lines tested as well as in two separate murine animal models. The change in detectable levels of the enzymes is not due to a change in amount of the SEAP enzyme present but instead is the result of a change in the activity of the SEAP enzyme.

Materials and methods

Gene constructs encoding wild-type and peptide conjugated proteins

All SEAP gene constructs were generated via direct PCR as previously described27.

In vitro DNA transfection via electroporation and reporter gene assays

Plasmid DNA was transfected into cells using electroporation in the following cell lines: EC40, C2C12, 4T1, Jurkat, and HEK293 (American Type Culture Collection, Manassas, VA). All cell lines were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (DMEM) (Life Technologies, Rockville, MD). Cells were then suspended at a concentration of 1×107 cells per milliliter of Opti-mem medium (Invitrogen, Carlsbad, CA) containing 10 µg/mL of firefly luciferase (Fluc) or Gaussia luciferase (Gluc) plasmid DNA (Valentis, Burlingame, CA, and New England Biolabs, Inc., Ipswich, MA, respectively) as transfection efficiency controls. 100 µL of the solution was transferred to individual electroporation cuvettes (n=3) to which 2 µg of experimental plasmid DNA was added. Each cuvette was pulsed with one 75 msec-pulse of 150 V. The cell suspensions and 900 µL DMEM were transferred to individual wells of 6-well plates, and the medium was collected and replaced on specified days. If needed, cell lysates were collected on the last day by incubating the cells with 200 µL of 1x Cell Lysis Buffer (Promega, Madison, WI), freezing the plates at −80 °C for 20 min, thawing the plates on ice, and collecting the lysate suspensions. All samples were stored at −80 °C until assayed.

Cell lysates and mediums were assayed for SEAP activity using the Phospha-Light SEAP Reporter Gene Assay System (Applied Biosystems, Foster City, CA) and following the manufacturer’s instructions. Fluc activity or Gluc acitivity was analyzed by assaying cell lysates with the Luciferase Assay System (Promega, Madison, WI) or cell mediums with the Gaussia Luciferase Assay System (New England Biolabs, Inc., Ipswich, MA) following the manufacturers’ instructions. Data is presented as fold comparisons of the concentrations of SEAP per Fluc or Gluc activity in the peptide-SEAP conjugates to the WT-SEAP.

Animal models, in vivo DNA delivery via electroporation, and serum collection

Six- to eight-week old Balb/c (in-house animal breeding facility) and nude mice (Charles River Laboratories, Inc., Wilmington, MA) were maintained under NIH guidelines approved by the Institutional Animal Care and Use Committee of Louisiana State University. Plasmid DNA, prepared as discussed above, was diluted in half-strength saline to 5 µg/30 µL, injected into each rear tibialis muscle, and directly followed by electroporation (n=4) as previously described28. The electroporation was performed using the following parameters: two 20 msec-pulses of 35 V/mm with a 100-msec interval between pulses. Blood was collected via cheek-bleeding on days 1, 5, 10, and 15 after transfection, placed on ice for 1 h, centrifuged at 3,000 × g for 10 min to separate the serum, and the serum was collected and stored at −80 °C until assayed as above.

Western blot analysis

10 µL of serum was subjected to SDS-PAGE in a 12% polyacrylamide gel and then transferred to a Trans-Blot Transfer Medium nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). Immunoblotting of the membrane was performed with a 1:500 dilution of the primary anti-Human Placental Alkaline Phosphatase polyclonal antibody (GeneTex, Inc., San Antonio, TX) and a 1:5000 dilution of the secondary anti-rabbit IgG antibody conjugated to horseradish peroxidase (GE Healthcare, Piscataway, NJ). The peroxidase signal was generated with the Western Lightning ECL (PerkinElmer, Waltham, MA) and visualized with a Kodak Image Station 440CF using the 1D Image Analysis Software v3.6 (NEN Life science Products, Boston, MA). All blots are representative of at least two repeat blots with similar results.

Statistical analyses

For all in vitro experiments, bars represent mean values of the SEAP/Fluc or Gluc activity divided by the mean WT-SEAP values, and the labels represent the fold-change compared to the control. For the in vivo experiments, the bars represent mean values of SEAP activity per microliter of serum divided by the mean WT-SEAP values. One-way ANOVA followed by Dunnett’s test was used for statistical analysis, and an asterisk (*) denotes p<0.05 comparing the SEAP-peptide mean value to the control SEAP mean value.

Results

Mini-peptide-coding sequences conjugated to SEAP plasmid DNA profoundly affect the activity of the resulting enzyme

Phage display technology has been widely used for in vivo biopanning to identify peptides that target tumors and other specific tissues29, 30. A group of peptides that were identified as tumor-targeted were analyzed for their ability to target tumors via insertion of mini-peptide-encoding segments directly before the stop codon on the C-terminus of SEAP plasmid DNA (Figure 1); however, in experiments performed in our lab the majority of the peptides failed to demonstrate any tumor targeting capabilities when fused with the SEAP reporter gene via a genetic engineering method (unpublished data).

Figure 1. Structure of wild-type (WT) and the peptide modified reporter gene plasmid DNA constructs.

Figure 1

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(a) The SEAP control construct. (b) Mini-peptide-SEAP fusion gene. Mini-peptide-coding sequences were directly inserted before the stop codon in the same open reading frame. CMV, Cytomegalovirus promoter; SEAP, secreted alkaline phosphatase; pA, polyadenylation signal; Stop, stop codon; Peptide, inserted peptide-encoding sequences.

Surprisingly, the different peptides caused drastic changes in the detected SEAP activity 24 h after transfection in EC40, an immortalized endothelial cell line. These changes ranged from more than a 3-fold increase compared to the wild-type SEAP (WT-SEAP) to almost undetectable activity (Figure 2a). There are some changes in the SEAP activities from cell lysates which correlates to the previously known tumor targeted peptides which are based on the RGD motif; however, there are no significantly different SEAP activities in the cell lysates (Figure 2b). So, the extreme changes in activity are not due to the peptides binding to the cell or the enzyme not being secreted from the cell. From this broad screening, we were able to conclude that the peptides that best enhanced the SEAP activity were CWDDWLC, TAASG, GSL, and RGD.

Figure 2. Differences in SEAP activity from transfection of endothelial cells with peptide-modified SEAP reporter fusion genes.

Figure 2

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The magnitude of secreted (a) and cell retained (b) SEAP activities from EC40 cells that were transfected with SEAP or SEAP fusion genes. Firefly luciferase (FLuc) plasmid DNA was co-transfected for normalization of the transfection efficiency variation. The abbreviations for each construct represent the single-letter amino acid code except for ‘WT’ which represents the wild-type SEAP activity. The bars represent the activity of SEAP (corrected for transfection efficiency by the activity of luciferase) using the wild-type SEAP/Luc activity to normalize the data. Bars and abbreviations in all subsequent figures represent the same data presentation unless otherwise noted.

To further investigate whether the increased activities by inserting these peptides are cell specific and whether insertion of these peptides results in a longer duration of increased expression, these selected peptide-SEAP fusion gene constructs were then transfected into murine muscle cells (C2C12), and the expression was determined for multiple days instead of just one day after the transfection. A plasmid DNA coding for Gaussia luciferase (Gluc), which is a secreted luciferase originating from the marine copepod Gaussia princeps31, was included in the transfection to correct for transfection efficiency. After transfection of these cells, the medium was collected and replaced on days 1, 2, 3, and 4 and analyzed for SEAP and Gluc activity (Figure 3a). The same as with EC40, one day after transfection the C2C12 cells produced significantly higher peptide-SEAP activities compared to the WT-SEAP with CWDDWLC nearing 3-fold higher activity and TAASG, GSL, and RGD at almost 2-fold higher activities. Furthermore, the fold-increases of CWDDWLC-SEAP compared to WT-SEAP on days 2, 3, and 4 were 2.51, 2.87, and 4.69, respectively. The other peptide-SEAP activities on days 2, 3, and 4 also remained higher than the WT-SEAP activities with RGD-SEAP elevating to 4-fold higher on day 4. Also, since the medium is replaced each day the increase in the activities is not from residual SEAP remaining from the previous day but new SEAP produced in a 24 h period. These data show that the elevated peptide-SEAP activities are not dependant on a specific cell type and results from newly produced enzyme units.

Figure 3. Increase in SEAP activity by inserting the selected peptides from murine muscle and tumor cells over time.

Figure 3

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Peptide-SEAP activity of the murine muscle cell line C2C12 (a) and adenocarcinoma cell line 4T1 (b) after transfection with peptide-SEAP constructs at each time point. The abbreviations are the same as described in the Figure 2 legend. An ‘*’ represents p<0.05 compared to the WT-SEAP activity.

Transfected murine tumor cells also produce increases in peptide-SEAP activities but to a much higher degree compared to the other tested cell types. When murine adenocarcinoma cells (4T1) were transfected in the same manner as the EC40 and C2C12 cells, all peptide-SEAP fusion protein reached greater activity with TAASG-, GSL-, and RGD-SEAP activities ranging from approximately 5- to 7-fold increases and CWDDWLC-SEAP having a greater than 11-fold increase in activity compared to the WT-SEAP on day 1 (Figure 3b). On days 2, 3, and 4 the peptide-SEAP activity continued to be higher but to a lesser degree compared to day 1 with only CWDDWLC- and TAASG-SEAP activities being significantly higher compared to the WT-SEAP on day 4. These results confirm that inserting peptide-encoding-sequences to the SEAP encoding sequence in the same open reading frame can have drastic effects on the resulting enzyme, and CWDDWLC is consistently the peptide that creates the highest elevation of SEAP activity.

Modification of the CWDDWLC sequence significantly alters the activity of the resulting enzyme

To further analyze whether the increase of SEAP activity by integrating the CWDDWLC-encoding DNA is dependent on the peptide sequence, we generated a construct containing an almost identical peptide with the insertion of one amino acid, yielding a sequence of CWDDGWLC-SEAP. The only difference in the peptides is a Gly residue located between the second Asp and Trp residues of the CWDDWLC peptide. We compared the activity of the new construct CWDDGWLC- with CWDDWLC- and WT-SEAP transfected into human cell lines.

Firstly, transfection of CWDDWLC-SEAP pDNA into human T (Jurkat), colon tumor (HT29), and kidney tumor (HEK293) cells resulted in elevated SEAP activity compared to the WT-SEAP similar to that seen in the murine cell lines (Figure 4). For both HT29 cells and HEK293 cells, the medium was collected the same as the murine cell lines so these data show that this phenomenon is not species specific and could translate into a clinical setting. Also, the Jurkat cell line is not adherent so instead of collecting and replacing the medium, 50 µL of medium was collected and assayed at every time point. The increase in CWDDWLC-SEAP activity occurs in all human cell lines derived from different tissues (Figure 4).

Figure 4. Increase in SEAP activity by inserting the peptide in human cell lines is amino-acid sequence specific.

Figure 4

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SEAP activity in Human T (Jurkat), colon tumor (HT29), and kidney (HEK293) cell lines on days 1 (a), and 4 (b). The abbreviations are the same as described in the Figure 2 legend. CWDDWLC, the selected peptide that maximizes the SEAP activity as was shown in Figure 3. CWDDGWLC, the sequence containing one Gly residue inserted in the CWDDWLC peptide to illustrate the sequence specificity. An ‘*’ represents p<0.05 compared to the WT-SEAP activity.

Secondly, the addition of the Gly residue dramatically and significantly diminished the detectable activity of the resulting SEAP enzyme (Figure 4). When the CWDDGWLC-SEAP DNA was transfected into all three human cell lines, the activity of the enzyme was at least10-fold less than the WT-SEAP activity and at least 20-fold less than the CWDDWLC-SEAP activity. So, the increase in activity is dependent on the structure and sequence of the CWDDWLC peptide.

Similar to the single amino acid insertion, we also tested amino acid deletion. Removal of the Cys residues that flank the peptide completely abolished the effect of the peptide on the SEAP activity (Figure 5). In both C2C12 and HT29 cell lines, transfection with the CWDDWLC-SEAP plasmid DNA repeatedly showed increased SEAP activity compared to the WT-SEAP transfected cells; however, Cys-less WDDWL-SEAP encoding plasmid DNA transfected cells produced levels of SEAP activity that were not significantly different than transfection with WT-SEAP encoding DNA (Figure 5).

Figure 5. Removal of the flanking Cys residues reduces the SEAP activity to the WT-SEAP level.

Figure 5

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SEAP activity in both human (HT29) and murine (C2C12) cell lines on days 1 (a) and 4 (b). CWDDWLC-SEAP, the reporter gene modified with the selected peptide coding sequence that maximizes SEAP activity. WDDWL-SEAP, the same selected peptide without the flanking Cys residues. An ‘*’ represents p<0.05 compared to the WT-SEAP activity.

The same trends in activity are seen with in vivo transfection but with no difference in the level of SEAP protein

After demonstrating that the increased activity seen by conjugating CWDDWLC to SEAP plasmid DNA occurs in all tested cell lines, the next step was to see if this effect also occurs in vivo, which determines the ultimate application value for reporting purposes. To determine this effect in vivo, mice were injected with 5 µg of peptide-SEAP plasmid DNA or WT-SEAP followed by electroporation in each rear tibialis muscle.

The same as seen in vitro, the CWWDWLC-SEAP activity was near 6-fold higher than that of the WT-SEAP activity on days 1 and 5 (Figure 6). Interestingly, the WDDWL-SEAP activity did appear to be increased on day 1, but this increase was not statistically significant, and by day 5 it was relatively the same as the WT-SEAP activity. Likewise, the CWDDGWLC-SEAP activity, while still significantly lower than the WT-SEAP, was only about 4-fold less than the WT-SEAP as compared to 10-fold less as seen in the in vitro data; however, by day 5 the CWDDGWLC-SEAP activity was undetectable in the serum. A similar experiment performed using nude mice treated with either CWDDWLC-SEAP or WT-SEAP plasmid DNA revealed similar results with differences in SEAP activity in the serum (data not shown).

Figure 6. Changes in peptide-SEAP activity from in vivo transfections.

Figure 6

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Mice were injected with 5 µg of plasmid DNA into each rear tibialis muscle followed by electroporation, and serum was collected and analyzed for SEAP activity on days 1 (a) and 5 (b). An ‘*’ represents p<0.05 compared to the WT-SEAP activity.

To determine if the changes in activity result from differences in the concentration of the SEAP protein, serum samples from each group on days 1 and 5 were analyzed via western blot using an antibody that is specific for the SEAP protein and does not bind endogenous mouse alkaline phosphatases. Astonishingly, the western blot revealed that there is no correlation between the changes in activity from the peptide-SEAP constructs and actual levels of the SEAP protein on any of the days tested (Figure 7). Even on day 5 when there was an almost undetectable level of CWDDGWLC-SEAP activity, there is no difference among the amounts of SEAP protein present in any of the groups.

Figure 7. Serum SEAP activity levels do not correspond to changes in the amount of SEAP protein present in the serum.

Figure 7

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The same serum samples assayed in Figure 6 were analyzed via western blot, and there were no differences in the amount of the SEAP protein present for WT-SEAP (a), CWDDWLC-SEAP (b), CWDDGWLC-SEAP (c), and WDDWL-SEAP (d).

Discussion

For the first time we show the dramatic effects that small peptides can have on the activity of SEAP due to integration of mini-coding-peptide sequences into the SEAP coding sequence in the same open reading frame. These peptides were believed to have tumor- and tissue-targeting abilities29, but the majority failed to show any targeting abilities in experiments performed in our lab (unpublished data). This result does not mean that these peptides do not have such tumor-targeted property, but instead it suggests that the peptides have to be meticulously used for the purpose of tumor targeting. Genetically engineering these peptides with this reporter gene seems to not be the best approach to exhibit the tumor-targeted property of these peptides, but when some of these peptides are fused with other viral vector genes, tumor-tropism was improved18, 32, 33.

Interestingly, we found that the peptides had extreme effects on the activity of SEAP reporter gene to which they were fused in the same open reading frame (Figure 2– Figure 5). Several methods are currently employed to increase the levels of gene products from the in vitro and in vivo delivery of DNA. Increasing the level of gene delivery is one area of focus for increasing the products of gene therapy. To accomplish this feat, several different modalities are used including the modification of viral vectors to elongate circulation times, reduce immunogenicity, and increase the accumulation of viral particles at target areas15, 17, 18. Also, the level of gene transfection with plasmid DNA can be increased by using several different techniques such as gene-gun, electroporation, ultrasound, and several others16. Likewise, increasing the level and duration of transgene expression can be accomplished by other specific methods: intuitive promoter selection8, 19–22, decreased unmethylated CpG levels6, 23, optimized codon use24, 25, and decreased size of transfected DNA26. Another manner is to increase the activity per molecule of gene products which has been accomplished via DNA shuffling for green fluorescent protein34. DNA shuffling and the new method reported here for increasing the activity of gene products has the potential to work in concert with these other widely used methods to further increase the efficacy of gene therapy. This concept should be tested in the future since it may improve the sensitivity of this reporter gene.

The increase in activity by insertion of the discovered peptide occurs in a gene-specific manner because the same peptide that enhances SEAP activity only slightly increases the expression of MHCI, a hallmark indicator of IFNa biological activity35, by CWDDWLC-IFNα compared to wild-type IFNa (data not shown). The increase was neither statistically significant nor therapeutically relevant. So, the large increase in activity seen in SEAP by the CWDDWLC peptide is not universal; however, other peptides may increase the biological activity of different therapeutic proteins. Screening several different peptides for individual proteins is necessary to discover the most effective peptide-protein combinations, which is the central concept for this MS. This concept is also different from the DNA shuffling technique in that the recombinant protein from DNA shuffling is generated through using variants of the same gene from the same and/or different species36 while our concept is to insert a peptide at the N or C terminal to increase the protein function.

One possible explanation for these dramatic differences would be that the peptides are interacting with cell surface ligands. Several of these peptides were thought to be targeted to the upregulation of certain proteins on the surface of cells during neovascularization due to tumor development4, 5, 37–39. Another explanation could be that the peptides somehow affected the secretion signal in the SEAP protein so the SEAP remained in the cell. However, these differences were not a result of peptide interactions with cell surface motifs or a loss of a secretion signal because the cell lysates did not show any significant differences in SEAP activity between the WT-SEAP and any peptide-SEAP groups (Figure 2b). Also, this change in activity was not dependent on the type of cell because the same differences were seen when murine muscle cells (C2C12) and adenocarcinoma cells (4T1) were transfected with the same SEAP constructs (Figures 3a, b, respectively). Likewise, this effect is also not restricted to murine cells because the same effects are evident in human immune (Jurkat), colon tumor (HT29), and kidney tumor (HEK293) cell lines (Figure 4). The varying activity levels of SEAP seen in the different cell types can be attributed to the cell-dependent activity of the CMV promoter. While CMV is a broadly used promoter for different cell lines and animal models, it is not always the best choice for all cell types8, 19, 21, 22, and modulation of the promoter based on the tissue to be transfected could lead to higher expression levels which will further increase the level of activity of the CWDDWLC-SEAP. So, the effects that the peptides have on the SEAP activity levels are not restricted to a certain cell type nor are they specific to a certain species. These facts are very important for the translation of the use of these highly sensitive reporter genes into other models such as large animals.

The sequence of the CWDDWLC peptide is very important for the increased activity effect to occur. Modification of the sequence by introducing a Gly residue between the second Asp and Trp residues rendered the resulting enzyme almost inactive (Figure 4). Further modification of the sequence by removing the flanking Cys residues also had serious consequences to the activity of the resulting SEAP enzyme. Interestingly, the loss of the Cys residues reduced the activity of the SEAP to almost identical the activity of the WT-SEAP enzyme (Figure 5). Interestingly, peptides with flanking Cys residues typically form loop secondary structures resulting from a disulfide bond between the two residues40. So, one possible explanation for the loss of the increase in activity could be due to the loss of the loop structure.

Another important aspect of this phenomenon is that it also occurs in vivo. As seen by measuring the SEAP activity in the serum of mice treated with the WT-SEAP, CWDDWLC-SEAP, and both modified peptide-SEAP constructs, the increase in CWDDWLC-SEAP activity as compared to the WT-SEAP continues for at least 5 days in both Balb/c (Figure 6) and nude mice. By days 10 and 15, the SEAP activity in all groups is reduced to insignificant levels (data not shown), which is most likely the result of an immune response against SEAP which is an enzyme of human origin. Amazingly the level of SEAP protein present in the serum of these mice is the same regardless of the activity of the different SEAP enzymes (Figure 7). So, the differences in the detectable levels of the SEAP enzymes are due to changes in the activity of the enzymes and not differences in the amounts of SEAP produced.

One of the main restraints which continues to hinder gene therapy is the level of gene expression, and several methods attempt to break these chains that bind gene therapy. There continues to be progress in this field, and the simultaneous use of multiple methods to increase the efficacy of gene therapy is important for gene therapy to become a major element in biomedical research and therapeutics. The use of peptides to increase the activity of proteins to which they are conjugated has the potential to be another method to synergistically increase the efficacy of gene therapy.

Acknowledgments

This work was supported by NIH/NCI grant RO1CA120895 and NIH/NIBIB grant R21EB007208.

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