Contents 1 Background 2 Introduction 2.1 TEG 2.2 GD5 2.3 Construction 3 Mechanism 3.1 Mechanism of A-B toxin infection 3.2 Chimeric Fusion Protein Facilitates Gene Transfer 4 Purification 4.1 Ni Column 4.1.1 Materials 4.1.2 Protocol 4.2 Modified protocol 4.2.1 Buffers 4.2.2 Protocol 5 Transfection 6 Results 6.1 Protein extract 6.2 Dialysis results 6.3 Plasmid 6.4 Primer 6.5 Biobrick 6.6 Adding system 7 Prospects 8 References

Background

Though therapeutic techniques are developing rapidly nowadays, some human diseases such as cancer and AIDS are extremely difficult to effect a radical cure. Gene therapy cures diseases by using DNA that encodes a functional, therapeutic gene to replace a mutated gene. Therefore, gene therapy is efficient to cure diseases results from gene mutation such as cancer and chronic infectious diseases.

Two major methods are applied to gene therapy. One is viral vectors. The other is non-viral vectors. Viral vectors are efficient to transfer foreign gene into cells and gene is efficiently expressed. But, on the other hand, viral vectors are hard to target specific cells which will decrease the percentage of the target gene into target cells. Moreover, we can't sure that viral vectors are not infectious. It ceases to be a safe way for gene therapy. As a consequence, non-viral vectors gain increasing attention since the late 20th century. Several methods for gene therapy with non-viral vector are studied by researchers such as electroporation, gene gun, magnetofection and so on.

Introduction

Our novel modular carrier protein is made up of three domains: Cell Binding Domain-specific binding to target cell, Translocation Domain- facilitating gene delivery via receptor-mediated endocytosis, DNA Binding Domain-specific binding to DNA containing UAS. Therefore, it can act as non-viral nucleic acid transfer system by delivering the carried target DNA to recognized the target cell via receptor-mediated endocytosis.

TEG

Novel DNA carrier protein TEG mimics the structure of Pseudomonas exotoxin A(ETA). TEG acts as a vector transferring specific target DNA into cells via receptor-mediated endocytosis. ETA consists of two subunits linked by disulfide bridge known as A-B toxin falling into three main structural and functional domains: the N-terminal receptor (R) binding domain I, translocation (T) domain II and the catalytic (C) domain III. Fig. 1 is the 3D structure of ETA.

The N-terminal receptor binding domain is replaced by a human EGF(epidermal growth factor) receptor ligand TGF-a. EGF receptor is overexpressed by human carcinomas cells. TEG containing TGF-a recognizes the EGF receptor and then binds to the EGF receptor. As a result, cells overexpressing EGF receptor are specifically combined with TEG. Similarly, replacing the C-terminal enzymatic domain of the toxin with the DNA-binding domain of the yeast GAL4 transcription factor leads to interaction with plasmid DNA of TEG. Overall, TEG contains TGF-a at N-terminal following by translocation domain of ETA and GAL4 at C-terminal

GD5

GD5 is another novel DNA carrier protein mimics the structure of diphtheria toxin (DT). DNA can be transferred into cells by GD5 via receptor-mediated endocytosis. DT is composed of two disulfide bridges linked subunits divided into three main structural and functional domains. The structure and function of DT are similar to ETA. But DT with cell binding domain at C-terminal and catalytic doamin at N-terminal, which is the inverse of ETA. Fig. 2 shows the 3D structure of GD5.

Similarly to TEG, chimeric fusion protein GD5 is assembled with antibody fragment specific for the tumor-associated ErbB2 antigen, translocation domain of DT as an endosome escape activity and Gal4 as DNA binding domain. Accordingly, ErbB2 antigen single chain antibody fragment FRP5 is placed at C-terminal via DT translocation domain, and GAL4 at N-terminal.

Construction

DNA fragments encoding amino acids 1 to 50 of human TGF-a, amino acids 252 to 366 of Pseudomonas exotoxin A (translocation domain) and amino acids 2 to 147 of the yeast GAL4 protein (DNA-binding domain) were assembled into one single open reading frame. The resulting plasmid pWF47-TEG encodes under the control of the IPTG-inducible tac promoter. A cluster of six histidine residues are added between TGF-a and ETA to facilitate the purification of the fusion protein via Ni2+ affinity chromatography. An N-terminal E. coli ompA signal peptide, a synthetic FLAG epitope for detection,and a KDEL signal for intracellular routing and full activity of TEG are the rest part of the chimeric fusion protein. The plasmid is under the control of the IPTG inducible tac promoter. Fig. 3 shows Schematic representation of the TEG fusion gene.

The construction of DNA carrier protein GD5 is similar to TEG, but GD5 does not contains ompA and KDEL. Fig. 4 shows Schematic representation of the GD5 fusion gene.

Mechanism

Mechanism of A-B toxin infection

A-B toxin infect human cell by binding specific cells and then translocate enzymatic domain into cells. They damage the cells by ADP-ribosylation-the transfer of ADP-ribose from NAD to a target protein, changes the behavior of the target protein. Here Figures. 5 shows the infectious mechanism of ETA.

1. Following removal of a C terminal lysine residue by plasma carboxipeptidase, the toxin binds to its cell-surface receptor (CD91, also called α2MR/LRP)

2. The toxin is internalized mainly via clathrin-coated pits

3. In the early endosome (EE), the toxin undergoes conformational change and is cleaved by the protease furin in a furin sensitive loop, in domain II. The two cleavage products remain linked by the intradomain disulfide bond

4. Following reduction of the disulfide bond, the enzymatically active C terminal fragment, which is composed of domain III and about two thirds of domain II, is routed to the trans-Golgi network where it binds via its C terminally exposed REDL sequence to KDEL receptor and travels to the endoplasmic reticulum (ER)

5. In the ER, sequences in domain II mediate the retro‑translocation of the polypeptide via the Sec61p translocon into the cytoplasm

6. The catalytic domain inactivates eukaryotic translation elongation factor 2 (eEF2) by ADP‑ribosylation, which causes translation inhibition and consequently cell death.

Chimeric Fusion Protein Facilitates Gene Transfer

The translocation domain of the chimeric fusion protein has function and mechanism similarly to the parental toxin. Hence, TEG or GD5 facilitates endosome escape of protein-DNA complexes upon internalization into target cells. Because of this property,an acidic environment is needed on the transit. Acidotropic reagent chloroquine have an enhancement of the efficiency of chimeric protein DNA delivery via receptor-mediated endocytosis. Endosomal acidification is blocked in the presence of chloroquine. Chloroquine but also result in endosome destabilization and the release of internalized DNA by accumulating in intracellular vesicles and inducing osmotic swelling of the endosomes.

Plasmids DNA is negative charged, which will do harm to the cell membrane when being transferring into cells. Moreover, it is not easy for plasmids DNA binding to chimeric fusion protein if they are scattered. Thereby, poly-cation interacts to DNA could neutralize the negative charged of plasmids DNA. And it can also condense DNA. As a consequence, more Plasmids can interact with chimeric fusion protein. In our project, poly-l-lysine acts as compensation of excess negative charged and condensation of DNA.

Purification

Ni Column

Materials

Binding buffer: 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 µM ZnCl2, 0.3 mM PMSF, 8 M urea, 10 mM imidazole

Wash buffer: 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 µM ZnCl2, 0.3 mM PMSF, 8 M urea, 50mM imidazole

Elution buffer: 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 µM ZnCl2, 0.3 mM PMSF, 8 M urea, 250 mM imidazole

Cleansing buffer: 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 µM ZnCl2, 0.3 mM PMSF, 8 M urea, 1 M imidazole

Protocol

1.Resuspend the bead s and add 15ml ddH2O to per-wash

2.Add 15ml binding buffer to balance the column and flow out all the binding buffer

3.Close the flow valve , add 15ml supernatant to the column, incubate for 30min, stir the beads when the beads precipitate.

4.Wash with 5ml binding buffer

5.Wash with 10ml wash buffer

6.Close the flow valve, add 1ml elution buffer, stir by pipetting, incubate for 2min, collect the eluent

7.Repeat step 6 three times

8.Wash with 15ml cleasing buffer

9.Wash with 20ml 22% ethanol

10.Store in 3ml 22% ethanol

Modified protocol

Buffers

Lysis buffer: 50 mM Tris–HCl pH 8.0,1 mM MgCl2,0.4 mg/ml DNase I, 0.4 mg/ml RNase A,1 mg/ml lysozyme.
Wash buffer 1: 20 mM Tris–HCl pH 8.0, 23% (w/v) sucrose,0.5 %(v/v) Triton X-100, 1 mM EDTA.
Wash buffer 2: 20 mM Tris–HCl pH 8.0, 1 mM EDTA.
Solubilization buffer: 6 M Guanidine HCl(we did not have it so we use 8M urea), 50 mM Tris–HClpH 8.0, 1 mM DTT.

Protocol

1. The steps before harvest the bacteria is the same as before

https://2014.igem.org/Team:SUSTC-Shenzhen/Notebook/A-B_Toxin/Purify#2014.8.25(steps before the step9 will be repeated by this protocol)

1. Suspend the pellet in 30 ml of lysis buffer, incubate it at 37°C

for 10–15 min, and sonicate it with a large sonicator tip.

1. Harvest inclusion bodies by centrifuge (25,000 × g for

30 min).

1. Wash inclusion bodies with wash buffer 1 by sonication, cen-

trifuge at 25,000 × g for 15 min and discard the supernatant. Repeat this step for five times.

1. Wash inclusion bodies with wash buffer 2 by sonication,

centrifuge and discard the supernatant.

1. Resuspend inclusion bodies in solubilization buffer, stir at

room temperature for 1–2 h.

1. If the gel result is ideal enough, we can ignore the steps of purification of Ni Column, which will decrease the loss of protein in the purify processes.

https://2014.igem.org/Team:SUSTC-Shenzhen/Notebook/A-B_Toxin/Modified_Protocol

Transfection

See the notebook of transfection

Results

Protein extract

Through our experiments, we successfully extract the protein, and can keep its concentration

Fig.6 we can see that in the 69kDa, there are band in the elution2,3,4 sample, it means, that the protein was presented by the bacteria. But the color of bands are not so deep, so the concentration may not reach our requirement.

Fig.7 similar to fig.6 , but the TEG protein is in the band of 43kDa, the color of TEG looks more deeper than GD5, but there are bands look like GD5 in the TEG protein. May be when we use the ddH2O to collect the plasmid on the letter, the TEG plasmid is mixed with few GD5 plasmid.

Dialysis results

And we use the dialysis to refold the protein, but failure. To test the DNA-protein binding activity, we use the NATIVE-PAGE

Fig.8 have no band, which means that there is no drift between DNA and this protein, maybe our protein did not refold successful.

Then, we changed our groups, make it more scientific

Plasmid

Primer

Biobrick

Adding system

Prospects

Chimeric fusion protein mimicking the structure of A-B toxin working as non-viral vector for gene therapy still has much room for development. These methods could be improved in many ways.

1. More than 90% of proteins are lost during the purification for the reason that after flowing through the Ni2+ affinity chromatography, protein weakly binding to Ni2+ will be washed before elution and only small percentages of denatured proteins can be successfully refolding. Methods purifying and refolding proteins should be improved, otherwise, it is difficult to apply to the clinic.

2. Chloroquine as acidotropic reagent has side effects of transfection. When cells are prolonged exposure chloroquine,cell viability will be affected or it will inhibit the proliferation of cells. According to the data published by Wels in 1998, chloroquine effects only 2 fold efficiency than transferring with chimeric protein alone. As a result, new acidotopic reagent can be found to enhance the efficiency.

3. Cell culture condition has influence on both cells and the chimeric protein. Optimal cell culture condition should be explored.

4. The chimeric fusion protein could be strengthened. Replacing more specific and binding affinity celling binding domain, or more efficient DNA binding domain is feasible. In addition, we can add more than one cell binding domain or DNA binding domain to enhance binding rate. Translocation domain from other A-B toxin should be applied to test the most efficient translocation domain.

A-B toxin basic non-viral gene transfer vector have a bright future if the efficiency is improved with the advantages that it is relatively safe and more specific than viral vectors.

References

[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1242130/pdf/bcr1264.pdf von Minckwitz, G., Harder, S., Hövelmann, S., Jäger, E., Al-Batran, S. E., Loibl, S., ... & Wels, W. S. (2005). Phase I clinical study of the recombinant antibody toxin scFv (FRP5)-ETA specific for the ErbB2/HER2 receptor in patients with advanced solid malignomas. Breast Cancer Research, 7(5), R617.]

[http://www.freepatentsonline.com/6498233.html Wels, W., & Fominaya, J. (2002). Nucleic acid transfer system.]

[http://europepmc.org/abstract/MED/9614577 Fominaya, J., Uherek, C., & Wels, W. (1998). A chimeric fusion protein containing transforming growth factor-alpha mediates gene transfer via binding to the EGF receptor. Gene therapy, 5(4), 521-530.]

[http://www.jbc.org/content/273/15/8835.short Uherek, C., Fominaya, J., & Wels, W. (1998). A modular DNA carrier protein based on the structure of diphtheria toxin mediates target cell-specific gene delivery. Journal of Biological Chemistry, 273(15), 8835-8841.]

[http://www.mdpi.com/2072-6651/2/11/2519/pdf Shapira, A., & Benhar, I. (2010). Toxin-based therapeutic approaches. Toxins, 2(11), 2519-2583.]

http://textbookofbacteriology.net/diphtheria_3.html