Development of a Protein Therapeutic That Targets the TMPRSS2:ERG4 Break Region in Prostate Cancer Cells: A Modular Design Approach ()
1. Introduction
Zinc finger (ZF) proteins account for 3% of the genes found in the human genome and have been shown to have roles in DNA replication, RNA packaging, transcriptional activation, regulation of apoptosis, protein folding and assembly, and lipid binding [1]. ZF motifs, or domains, are independently folded domains found within ZF proteins and contain a two-stranded antiparallel β-sheet as a β hairpin, an α helix and a zinc (II) ion. The zinc ion is tetrahedrally coordinated between the two cysteines (at the turn in the β hairpin) and two histidines (in the C-terminal part of the α helix) to form an inner hydrophobic core (X7-Cys-X4-Cys-X12-His-X3-His-Xn).
The ZF protein binds to its target DNA sequence by slotting the α helix into the major groove of the DNA. Each ZF domain recognizes, with a varying degree of specificity, a three-base segment on one strand of the DNA. Partners of many different ZF proteins have been determined [2]. Our therapeutic was designed to make use of several ZF domains that, when covalently linked, would be specific to the prostate cancer gene TMPRSS2:ERG4 [3] [4]. The ZF domains can be selected through the use of ZiFit, a program that determines ZF binding partners to specific sequences of DNA.
Multiple ZF domains have been shown to produce a greater degree of discrimination and complexity of binding to a specific DNA sequence [5] [6]. Therefore, the use of several ZF domains in tandem can promote tight, nearly (or completely) irreversible binding to specific DNA sequences. ZiFit does give several selections for each three-base sequence; our aim was to perform parallel binding studies to determine which combination of ZF domains are the most effective in binding to the TMPRSS2:ERG4 gene, as covalently linking two independent ZF domains can result in change in ability to predict binding to the DNA target [7]. We began by focusing on the use of five ZF proteins overlapping the breakpoint region, covalently bound together by short flexible linkers.
Human Uracil DNA Glycosylase, isoform 2 (UDG2) is a protein enzyme involved in the removal of uracil from DNA as part of the cell’s DNA repair mechanism [8]. Two forms of UDG are generated from the UNG gene—the mitochondrial UDG, UDG1, which consists of 304 amino acids, and the nuclear UDG, UDG2, which consists of 315 amino acids. Roughly 90% sequence homology exists between the two isoforms; the difference at the N-terminus is for cellular localization, not for catalytic activity [9] [10]. We used UDG2 in this study, as we wished to use its nuclear localization sequence (NLS) to target the chromosomal DNA. The mechanism of UDG2 interaction with DNA appears to be dominated by DNA hopping between sequences of approximately 10 bp; fast 1-dimensional scanning along this length of sequence takes place before the enzyme hops to another segment of DNA in search of the specific uracil lesion to which it has high affinity. This nonspecific binding of UDG exhibits a very short residence time of approximately 5 ms [11]. The processivity of the E. coli form of UDG is affected by NaCl concentrations [12]. The enzyme must successfully bypass all of the potential blocks that it encounters, including transcription factors and nucleosome remodeling.
GATA proteins are a family of transcription factors that bind, via one or more ZF motifs, to a canonical GATA DNA sequence found at promoter regions of key regulatory proteins [13] [14]. In addition to the ZF motifs, which are primarily responsible for the GATA binding to GATA DNA sequences, wild-type GATA proteins have a lysine-rich C-terminal tail that has been shown to be necessary for maintaining high DNA binding affinities [15] [16].
It has been reported that there are 358 gene fusions known to cause tumorigenesis, which are accountable for 20% of the human cancer morbidity [17]-[20]. Translocation of chromosomal DNA causes rearrangements of genetic material that can lead to the formation of oncogenes. Most prostate cancer cells have DNA that has been cleaved and rejoined in such a way as to create a breakpoint region. Cancer caused by this manipulation of DNA stems from the fact that the growth regulation factors of the cell are damaged or removed; this leads to unregulated cell growth and lifetimes [21] [22]. The effects of this alteration of the cellular DNA could potentially interfere with a number of biological systems, including (but not limited to) overexpression of specific proteins, alteration of apoptotic pathways (preventing programmed cell death), or cell growth (stimulating unregulated cellular expansion). The breakpoint region that is produced by the co-joining of two different genes is typically a unique sequence not found in normal cells. This provides an opportunity for drug targeting to this region of genomic DNA that is specific to prostate cancer cells only, leaving normal cells unaffected.
2. Materials and Methods
2.1. Expression Vector
Figure 1. Complete DNA sequence for the Prostate Cancer Therapeutic. This gene includes sequences for Uracil DNA Glycosylase and five Zinc-Finger Protein (ZFP) domains, as well as the two types of linkers that connect them.
The gene sequence for our proposed prostate cancer therapeutic (PCT) was synthesized and cloned into the pET-24a(+) vector system using the BamHI and NdeI cloning sites by Twist Bioscience. This system is an overexpressing construct containing the kanamycin resistance gene. Codon optimization was carried out for E. coli codon usage using the Twist Bioscience sequence optimization software. Verification of the gene insert was carried out using Sanger sequencing (Twist Bioscience). Sequence data showed that the gene had the correct restriction sites and the gene sequence was correct. The full DNA sequence for the PCT can be seen in Figure 1; Figure 2 shows the protein sequence of the entire designed therapeutic (54.54 kDa calculated molecular weight; bioinformatics.org).
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Figure 2. Protein sequence for the Prostate Cancer Therapeutic (PCT). Amino acids are color-coded as follows: Uracil DNA Glycosylase (UDG)—black letters; Zinc-Finger (ZF) domains—green letters; SR linkers—red letters; Bridge-linkers/basic tail sequences—orange letters.
2.2. Transformation of PCT Gene Construct
BL21 (DE3) cells (Invitrogen) were transformed by adding 1.0 ng of the pET 24a(+) vector containing the gene sequence for PCT. Cells were then heat-shocked as follows: Cells were incubated on ice for 1 hour, placed in a 40˚C hot water bath for 30 s, then immediately placed on ice for 5 min. 500 μL of 37˚C SOC media (National Diagnostics) was added and incubated at 37˚C for 1 h. The cells were spun down and spread out on a LB-agar plate containing the kanamycin antibiotic. The plate was incubated overnight at 37˚C.
2.3. Isopropyl β-D-1-Thiogalactopyranosid (IPTG) Induction
A single colony was picked and inoculated in 5 mL of LB media containing 50 μg/mL kanamycin in a 15-mL conical tube. The resulting solution was incubated overnight at 37˚C in a shaking incubator. Cells were diluted 1:100 in 200 mL LB media containing 50 μg/mL kanamycin and grown at 37˚C in a shaking incubator until the optical density at 600 nm reached 0.6 OD units. At this point, 1 mM IPTG was added and the cells were left to shake for 3 h before being removed. The cell suspension was centrifuged at 3000×g for 30 min at 4˚C. The cell pellet was then suspended in liquid nitrogen for 1 min, then placed in a −80˚C freezer for storage.
2.4. Protein Expression and Purification
PCT proteins were isolated from crude cellular extracts by resuspending 10 g of wet cell pellets in 40 mL metal chelate (MC) binding buffer solution (5 mM imidazole, 250 mM NaCl, and 20 mM Tris-HCl (pH 7.9). A lysis buffer was made by adding 0.1% NP-40 (to promote solubility), 0.5 mM PMSF, and 1.0 mM benzamidine (to inhibit any endogenous proteases) to the 40 mL MC buffer containing the resuspended cells. Cells were lysed by subjecting them to a single round of a freeze/thaw cycle (−20˚C for 24 h), followed by 10 repetitions of a 10-s sonication with a 1-min cooling cycle on ice. Sonicated cell extracts were then centrifuged at 3000×g for 40 min at 4˚C. The supernatant containing the PCT proteins was collected and applied to a 7-mL Nickel-NTA HisPurTM Econo column (Bio Rad/Thermo Scientific). Prior to loading the cell extract, the affinity column was equilibrated in the MC metal chelate-binding buffer. The supernatant was loaded onto the column at a flow rate of 1 mL/min. The column was washed with 4 column volumes of MC buffer before starting the elution step. The bound protein was eluted over 3 column volumes of metal chelate elution buffer (1 M imidazole, 500 mM NaCl, and 20 mM Tris-HCl (pH 7.9)). Fractions were analyzed on 12% SDS-PAGE gels. Each protein eluted as a single peak within 3 - 5 fractions at an imidazole concentration of approximately 350 mM.
2.5. Protein Identification by Liquid Chromatography-Tandem
Mass Spectroscopy (LC-MS/MS)
LC-MS/MS was performed using the same conditions as previously described [23]. Analysis was performed by the Proteomics Department at the University of Vermont (Burlington, VT).
3. Results
Our results show a proof of concept for the modular design of a protein cancer therapeutic.
3.1. Drug Design and Process
3.1.1. Building a Specific Zinc Finger Array for Binding to the
TMPRSS2:ERG4 Gene
The Zinc Finger Consortium (http://www.zincfingers.org/default2.htm) has studied binding of individual ZF modules to their target DNA sequences [2] [24]. The ZiFiT (Zinc Finger Targeter) program [25] [26], which has been developed by the Consortium, facilitates the design of custom zinc finger proteins (ZFPs) by identifying suitable DNA target sites and matching them with validated zinc finger modules using a modular assembly approach. We utilized ZiFiT’s “Modular Assembly” method, which aims to construct ZFPs by linking three individual pre-characterized ZF modules, each recognizing three consecutive and specific 3-bp DNA subsites, together. The ZiFiT program accepts as input uploaded DNA sequences, which it then divides into 9-bp regions composed of three consecutive 3-bp triplets. It then compares each triplet of triplets against a library of experimentally-validated ZF modules. Because three ZF proteins are required to bind to 9-bp regions, the selectivity of the designed ZFP system is increased and the chance of off-target binding is decreased. Only compatible triplets (especially GNNs) are retained for reliable design. Each potential 9-bp target site is evaluated based on GNN Score (preference is given to sites with more GNN triplets, as they yield higher functional success) and Affinity Score (a predictive metric indicating DNA-binding strength, where lower values suggest stronger binding and better functionality). Individual ZF–to–triplet affinities (e.g., a numeric Kd for a single ZF, such as ZF83 binding to AGC) have not been published or centrally curated, and these data are not reported in ZiFDB or Consortium notes. However, it has been reported that assembled multi-finger proteins typically bind with high affinity in the low nanomolar range (Kd ≈ 1 − 20 nM) and with high specificity [27].
For our study, we uploaded the TMPRSS2/ERG DNA sequence of interest into ZiFiT and selected target sites with high GNN content and favorable Affinity Scores. We selected five promising ZF proteins that should, when expressed as a covalently-linked system, bind with high specificity to DNA triplets found around the breakpoint region of the prostate cancer gene TMPRSS2:ERG4 [3] [4], specifically AGC (for the first zinc finger) and GCG (for the second) in the TMPRSS2 exon 1 portion of the breakpoint region, and AGG (for the third zinc finger), AAT (for the fourth) and CTT (for the fifth) on the ERG4 portion of the breakpoint region. Based on these results, we selected ZF83, ZF23, ZF84, ZF77 and ZF103 as modules for the ZF array using the ZF numbering introduced by the Zinc Finger Consortium (see Figure 3). Figure 4 shows a schematic for the overall concept for the design of the PCT protein.
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Figure 3. TMPRSS2/ERG DNA sequence. The TMPRSS2 sequence is shown in orange, and the ERG sequence is shown in blue. The red triangle indicates the break-region. Zinc Finger Proteins are shown at their respective triple base-pair binding sites (ZF numbering is from the Zinc-finger Consortium) and include ZF83 (AGC); ZF23 (GCG); ZF84 (AGG); ZF77 (AAT); and ZF103 (CTT).
Figure 4. Schematic showing the PCT protein overlapping the TMPRSS2/ERG4 break region. The image shows the ribbon structure of UDG linked to five Zinc-Finger Proteins (ZFP). Each ZFP is aligned along the DNA in their approximate positions with respect to the break region.
3.1.2. Molecular Motor Design
UDG2 has an NLS, which should be useful in vivo to locate the system to the chromosomal DNA in an organized fashion as opposed to the ZF array randomly encountering its target sequence through diffusion. UDG2 can also act as a molecular motor, pulling the system along the DNA in search of its target sequence. One of UDG’s functions is to unpackage, unwind and clear chromosomal DNA, which may assist in locating the system to the nucleus and increasing the accessibility of the DNA target. In addition, the presence of the UDG may serve to overcome nonspecific interactions of the zinc finger system to random DNA sequences, as the force of UDG hopping and short-range scanning may overcome any nonspecific, weaker interactions the ZF array may experience.
3.1.3. Linkers/Anchor Design
Wild-type GATA proteins have ZF motifs that are primarily responsible for binding to specific DNA sequences. They also contain a lysine-rich C-terminal tail (or anchor) that has been shown to be necessary for maintaining high DNA binding affinities [15] [16]. We predict that inclusion of a C-terminal tail found in the GATA1 protein (protein sequence DVIKKRNR) will improve the binding affinity of the UDG-containing fusion system to the TMPRSS2:ERG4 gene sequence. We have also used the DVIKKRNR sequence to bridge between sets of two zinc finger modules; each individual module is joined by short SR linkers (protein sequence SR). The fifth ZF motif is preceded by the longer DVIKKRNR sequence to introduce necessary flexibility for binding to the DNA target.
3.2. Protein Expression and Purification
3.2.1. Protein Expression
Protein expression was carried out according to the conditions outlined in Section 2.4. Colony growth on antibiotic-inclusive plates were excellent, with large, well-distributed colonies present after 12 - 15 h incubation. Figure 5 shows the expression profile for the induction of the protein therapeutic PCT. When comparing the pre- and post-induction lanes, we see that they share a number of similar-sized protein bands. However, the post-induction has an additional heavy band at approximately 40 kD that is not present in the pre-induction lane. These results are not consistent with the expected molecular weight of 54 kD for the PCT protein.
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Figure 5. Gel of expression for the PCT/pET24a(+) construct. PL = protein ladder (MW values are labeled to the left of the gel); Pre is pre-induction; Post is post-IPTG induction after 4 hrs. The black arrow points to the heavy band at approximately 40 KD in the post-induction lane that indicates the overexpression of the E. coli OmpF protein, which is verified by LC-MS/MS as shown in Table 1.
3.2.2. Affinity Chromatograph
Figure 6. Gel of the eluted protein from Nickel-NTA purification. PL indicates protein ladder (MW values are labeled to the left of the gel); EP is the eluted protein lane. The black arrow points towards the band at approximately 40 KD in the eluted protein sample lane (EP) that indicates overexpression and elution of the E. coli OmpF protein as verified by LC-MS/MS (see Table 1).
Protein extracts were purified using a Nickel-NTA column under the conditions outlined in Section 2.4. The eluted protein sample was visualized on a 12% SDS-PAGE gel (Figure 6). The only band present is at the 40 kD mark, which corresponds to the overexpressed protein in Figure 3. In order to determine if this was a truncated PCT or a different protein entirely, the gel piece containing the band was removed and analyzed by LC-MS/MS.
3.2.3. Protein Identification by Liquid Chromatography-Tandem Mass
Spectroscopy (LC-MS/MS)
From the LC-MS/MS data, it can be seen that the predominant protein found is an E. coli protein, Outer Membrane Porin F (Table 1). This protein was identified with high confidence and a high coverage (>10 peptides with a high peptide identification score). From the SDS-PAGE analysis, these results have been consistent throughout the overexpression optimization process for PCT. No PCT fragments were found in the LC-MS/MS results, indicating that protein expression of the target therapeutic was not successful.
Table 1. Sample Identification by Liquid Chromatography-Tandem Mass Spectroscopy (LC-MS/MS). The table shows the identity and information for the 25 most significant proteins detected from an excised protein sample as shown in Figure 6. The proteins are listed from the most to least abundant proteins in the sample analyzed.
Accession |
Description |
Coverage [%] |
#Unique Peptides |
#AAs |
MW [kDa] |
calc. pI |
Score Sequest HT: Sequest HT |
Q8XDF1 |
Outer membrane porin OmpF OS = Escherichia coli O157:H7 OX = 83334 GN = ompF PE = 3 SV = 1 |
31 |
11 |
362 |
39.3 |
4.96 |
70.55 |
P04264 |
Keratin, type II cytoskeletal 1 OS = Homo sapiens OX = 9606 GN = KRT1 PE = 1 SV = 6 |
19 |
11 |
644 |
66 |
8.12 |
32.95 |
P13645 |
Keratin, type I cytoskeletal 10 OS = Homo sapiens OX = 9606 GN = KRT10 PE = 1 SV = 6 |
19 |
10 |
584 |
58.8 |
5.21 |
30.64 |
P35908 |
Keratin, type II cytoskeletal 2 epidermal OS = Homo sapiens OX = 9606 GN = KRT2 PE = 1 SV = 2 |
20 |
8 |
639 |
65.4 |
8 |
30.02 |
P0A911 |
Outer membrane protein A OS = Escherichia coli O157:H7 OX = 83334 GN = ompA PE = 3 SV = 1 |
29 |
9 |
346 |
37.2 |
6.42 |
29.56 |
P35527 |
Keratin, type I cytoskeletal 9 OS = Homo sapiens OX = 9606 GN = KRT9 PE = 1 SV = 3 |
14 |
9 |
623 |
62 |
5.24 |
26.46 |
Q8XBD3 |
Outer membrane protein assembly factor BamC OS = Escherichia coli O157:H7 OX = 83334 GN = bamC PE = 3 SV =4 |
19 |
6 |
344 |
36.8 |
5.55 |
19.71 |
P0A6P3 |
Elongation factor Ts OS = Escherichia coli O157:H7 OX = 83334 GN = tsf PE = 3 SV = 2 |
21 |
6 |
283 |
30.4 |
5.29 |
13.32 |
A0A0H3JCB2 |
Lactose operon repressor OS=Escherichia coli O157:H7 OX = 83334 GN = lacI PE = 4 SV = 2 |
10 |
4 |
363 |
38.9 |
7.17 |
9.86 |
Q8XAA9 |
Outer membrane protein assembly factor BamB OS = Escherichia coli O157:H7 OX = 83334 GN = bamB PE = 3 SV = 1 |
13 |
4 |
392 |
41.8 |
4.91 |
9.83 |
P02533 |
Keratin, type I cytoskeletal 14 OS=Homo sapiens OX = 9606 GN = KRT14 PE = 1 SV = 4 |
6 |
2 |
472 |
51.5 |
5.16 |
8.8 |
P02538 |
Keratin, type II cytoskeletal 6A OS=Homo sapiens OX=9606 GN=KRT6A PE=1 SV=3 |
6 |
1 |
564 |
60 |
8 |
8.39 |
P65765 |
FKBP-type peptidyl-prolyl cis-trans isomerase FkpA OS = Escherichia coli O157:H7 OX = 83334 GN = fkpA PE = 3 SV = 1 |
8 |
2 |
270 |
28.9 |
8.47 |
7.7 |
P13647 |
Keratin, type II cytoskeletal 5 OS=Homo sapiens OX = 9606 GN = KRT5 PE = 1 SV = 3 |
5 |
1 |
590 |
62.3 |
7.74 |
6.67 |
P81605 |
Dermcidin OS = Homo sapiens OX = 9606 GN = DCD PE = 1 SV = 2 |
20 |
2 |
110 |
11.3 |
6.54 |
4.96 |
Q8X773 |
Uncharacterized protein OS=Escherichia coli O157:H7 OX = 83334 GN = ydgH PE = 4 SV = 1 |
9 |
2 |
314 |
33.9 |
9.28 |
4.63 |
P0A7Z6 |
DNA-directed RNA polymerase subunit alpha OS = Escherichia coli O157:H7 OX = 83334 GN = rpoA PE = 1 SV = 1 |
5 |
2 |
329 |
36.5 |
5.06 |
3.84 |
P0AA27 |
Thioredoxin 1 OS = Escherichia coli O157:H7 OX = 83334 GN = trxA PE = 1 SV = 2 |
11 |
1 |
109 |
11.8 |
4.88 |
3.37 |
Q86YZ3 |
Hornerin OS = Homo sapiens OX = 9606 GN = HRNR PE = 1 SV = 2 |
2 |
1 |
2850 |
282.2 |
10.04 |
2.83 |
E7EQ64 |
Trypsin-1 OS = Homo sapiens OX = 9606 GN = PRSS1 PE = 1 SV = 1 |
8 |
1 |
261 |
28.1 |
7.25 |
2.61 |
P69799 |
PTS system mannose-specific EIIAB component OS = Escherichia coli O157:H7 OX = 83334 GN = manX PE = 3 SV = 2 |
4 |
1 |
323 |
35 |
6.02 |
2.53 |
P0A9S4 |
Galactitol 1-phosphate 5-dehydrogenase OS = Escherichia coli O157:H7 OX = 83334 GN = gatD PE = 3 SV = 1 |
3 |
1 |
346 |
37.4 |
6.38 |
2.45 |
P0A9B4 |
Glyceraldehyde-3-phosphate dehydrogenase A OS = Escherichia coli O157:H7 OX = 83334 GN = gapA PE = 3 SV = 2 |
5 |
1 |
331 |
35.5 |
7.11 |
2.14 |
P0AAC2 |
Universal stress protein E OS=Escherichia coli O157:H7 OX = 83334 GN = uspE PE = 3 SV = 2 |
2 |
1 |
316 |
35.7 |
5.31 |
2.08 |
P58070 |
GTPase Era OS = Escherichia coli O157:H7 OX = 83334 GN = era PE = 3 SV = 1 |
2 |
1 |
301 |
33.7 |
7.65 |
2.07 |
4. Discussion
In this study, we designed a system that theoretically would recognize and bind to a specific breakpoint region of the prostate cancer gene TMPRSS2:ERG4 [20]. Fusion of the TMPRSS2 gene to the ETS transcription factor ERG has been reported in up to two-thirds of prostate cancer cases (for example, see [3] [4] [20] [28]-[33]). It has been shown that translocations of TMPRSS2 to ERG can result in at least 17 distinctly structured fusion transcripts [20] [31] [34], but the most commonly detected transcript involved joining of exon 1 of TMPRSS2 to exons 4 or 5 of the ERG gene [20]. This leads to overexpression of the 3’-exons of the ERG gene. We initially focused on the joining with exon 4 of the ERG gene (TMPRSS2:ERG4), as it has been suggested that this fusion is the most abundant in prostate cancer samples [30].
Our objective was to design a protein therapeutic that would suppress the growth of prostate cancer cells that contain TMPRSS2:ERG4. Since this breakpoint region is found only in prostate cancer cells, no healthy cells will be affected. The therapeutic consisted of a UDG2 component, an array of ZF protein modules, and a lysine tail. The array contained five covalently-linked ZF protein modules that were designed to bind with high specificity and affinity to the TMPRSS2:ERG4 translocation, would block replication and trigger cell death, thus preventing its proliferation. The UDG2 component N-terminal to the array would locate the system to the nucleus in an efficient fashion, where it would unwind the DNA from histones and clear it of bound proteins and small molecules (thus making the DNA accessible to ZF binding) while also acting as a molecular motor to pull the system along the chromosomal DNA in search of the TMPRSS2:ERG4 region. However, the force of motion of UDG2 along the DNA may be strong enough to overcome the high binding affinity of the designed array. Inclusion of a lysine-rich tail such as is found in zinc finger-containing GATA proteins was included to potentially help to balance the force of motion of the UDG2, thereby rescuing some or all of the binding affinity of the zinc finger system.
Other applications have used multiple ZF proteins fused to nonspecific nucleases for down-regulation of genes in the treatment of genetic disorders; in those applications, the zinc fingers locate the gene of interest and a fused nonspecific nuclease introduces a double-strand breakage at a specific site [35].
5. Conclusions
In this article, we present a novel drug design using a modular approach to target the TMPRSS2:ERG4 gene fusion [36] [37]. The data in this paper highlight the barriers encountered when attempting to combine the functions of proteins from different species in order to utilize their unique characteristics; our therapeutic included Uracil DNA glycosylase (Homo sapiens); a linker sequence (Aspergillus nidulans); and synthesized zinc-finger domains (predominantly derived from plant species by the Zinc Finger Consortium). Although every effort was made to generate an optimized gene sequence with a high degree of confidence, there may still be unforeseen issues in the algorithm we used in terms of sequence complexity that is unacceptable to the E. coli cells overexpressing the therapeutic, rare codon usage, or other factors.
Another reason why the expression of the drug was unsuccessful could be a leaky promoter. pET24a(+) vectors are known to express low level amounts of the gene pre-induction. It is conceivable that the low levels of expressed PCT could competitively bind at the promoter region, preventing further expression, even after induction with IPTG. Because this system was designed to bind to DNA, this would suggest that the drug binds indiscriminately—something that is highly unfortunate in the design of a therapeutic meant for interaction with a specific DNA sequence.
It is interesting that the overexpression of the Outer Membrane Porin F protein (OmpF) native to E. coli is reproducibly overexpressed; others have reported that expression of OmpF is downregulated under antibiotic stress [38] [39]. It is even more interesting that it is retained and detected at such high levels after metal-affinity column purification, as OmpF is not rich in histidine residues. However, it has been seen that BL21 (DE3) cell lines do have elevated levels of OmpF overexpression as compared to other E. coli strains [40] [41].
To address the lack of target PCT expression, future studies will include reducing the number of zinc-finger modules in varying arrangements to see the effects on protein expression. We are also in the process of examining the use of a eukaryotic vector-based system for protein expression in a mammalian cell line, which might be more tolerant of the protein therapeutic.
Acknowledgements
Research reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103449. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.
The authors wish to acknowledge the previous work of our Saint Michael’s College research students that, while not represented in this paper, did work on this project. These students include Sara Williams, Isaiah St. Pierre, Dana Bourne, Christopher Ricciardi, David Weiss, Megan Ackerman, Christopher Toomey, Samantha Delaney, and Tiana Dunne.