Design and assembly

We aim to ensure that the specifications that were drawn up are considered in the design. In recently studies, Apoptin has been described to induce apoptosis in various human cancer cell lines. To develop apoptin as an anti-cancer drug, efficient transduction tools have been used to deliver apoptin into tumor cells. cell-penetrating peptides, such as the trans-acting activator of transcription (TAT) protein transduction domain (PTD) and MPG peptide have therefore been used as vectors for protein delivery.So we decided apoptin fused to TAT peptide because this peptide move apoptin into cell without membrane damage.Also we fused Hemoglutinin A with TAT in order to decrease possibility of getting trapped in microcytosis.In addition to we conceived an alternative technology that offers fusing another cell penetrating peptide with apoptin, thus we constructed MPG peptide with apoptin.And we applied that idea again, on onether protein which names e4of4.E4of4 has been knew induce apoptosis as a tumor specific. We demonstrated e4of4 fused to TAT peptide as a againist tumor cells.As a result, we brought MPG-Apoptin and TAT-E4of4 to literature. For apply that project, we ordered apoptin fused to TAT, TAT HA and MPG sequences respectively as a composite part in order to minimalize cost and time.And we cloned into pSB1C3 backbone.After cloning, proteins were expressed in BL21 e. coli strain under constutive promoter firstly.İn addition to,our constructs were harboring a 6xHis tag.The 6×His were fused with constructs at the N-terminus because enhance purification.

After cultivatization, we controlled our genes in gel electrophoresis. Electrophoresis showed us the transformation of new genes was successful. There are gel electrophoresis results especially content of Module 2 below:

Experiments and results: To purify the recombinant proteins, cells were spun down from 50 mL of culture supernatant and resuspended in phosphate buffer saline.PBS contained PMSF in order to prevent proteis from proteases. The mixture was then sonicated on ice eight times for 30 second with a 52% pulsed activity cycle (MISONIX Sonicator W 300). Next, the lysate was centrifuged for 30 min at 10,000 rpm to remove the cell debris. The resulting cell supernatant was loaded onto HIS select affinity column (Sİgma Aldrich)) for protein purification using the standard procedure.. The total protein concentration of each collected fraction from the column was determined using Bradford protein assay with bovine serum albumin acting as the reference protein. The purity of the protein from each fraction was analyzed by 12.5% SDS-PAGE and then the resulting gels were Western blotted using monoclonal anti-HIS antibody.

Using western blot analysis, it was observed that; our proteins expressed and purified effectively. Firstly, We offered that using Immunofluorescence staining method for understanding TAT fusing proteins lokalization.Therefore,we fused to TAT-RFP construct with 6xHis tag in order to prove TAT activity.Immunofluorescence is a technique used for light microscopy with a fluorescence microscope and is used primarily on microbiological samples. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows visualisation of the distribution of the target molecule through the sample.When Flourochrome labeled anti Histag antibody bond 6His tag which in front of our construct we visualized TAT-RFP intracellular area.Also we stained DNA and nuclei using Hoechst dye (Thermo Scientific Pierce Hoechst 33342 Fluorescent Stain ) thus we were sure that, TAT peptide alllows penetration .

Investigate whether his-TAT-Apoptin, TAT-HA-Apoptin, TAT-E4of4 and MPG-Apoptin protein expressed by E. coli has apoptotic activity when intro-duced into tumor cells, purified protein was used to examine the protein's apoptotic activity when it was used to treat human colon cancer HT29, breast cancer MCF7 and head-neck cancer cell lines.while we used different factors such as dosage, cell line, changing purified methods we repeated experiments.so we analysed cell viability index under changing factors.We used xCelligence for analysis. The xCELLigence System, also known as RT-CES system, which contains a series of Real Time Cell Analyzer (RTCA), is a labeling free cell based assay system integrating microelectronics and cell biology, suitable for uninterrupted monitoring of biolgical processes of living cells. We conducted experiments on in vitro cultured HT29 cells which were treated with TAT-Apoptin, TAT-E4of4, TAT-HA-Apoptin and MPG-Apoptin. The results of the X-Cellingence revealed that our constructs was able to significantly inhibit the growth of the HT29 cells. We used only DMEM and elution buffer containing well as a control respectively.

In growth rate of HT 29 cells was performed by xCELLigence after %5 concentration proteins of the purified E. coli-expressed recombinant proteins was co-cultured with HT-29 cell for 54 hours. Non-apoptosis controls, only DMEM and only elution buffer treated cells were assayed. These represent respectively HL-60 cells only (blue), HT-29 cells co-cultured with protein elution buffer (brown) and HT-29 cells co-cultured with TAT-Apoptin (light green),TAT-HA-Apoptin (dark green), MPG-Apoptin (light blue) and TAT-E4of4(purple).

	This results observed that in a certain range of concentration and time, our proteins  inhibited the proliferation of the HT 29 cells time dependently. Its inhibitory effect was %5 concentration and 54 hours. 

Using a similar approach, we tested the apoptosis-inducing ability of our constructs in HEK 293 human kidney cell lines.HEK 293 were co-cultured with TAT-Apoptin, TAT-E4of4, TAT-HA-Apoptin and MPG-Apoptin.In a desired concetration; we expect that; growth curve of treated with our construct HEK 293 cells should be similary growth curve of not treated HEK 293.

Figure 1: In growth rate of HEK293 cells was performed by XCelligence after %5 concentration proteins of the purified E. coli-expressed recombinant proteins was co-cultured with HEK-293 cell for 54 hours. These represent respectively HEK-293 cells only (dark green )), HT-29 cells co-cultured with protein buffer ( blue) and HT-29 cells co-cultured with TAT-Apoptin (red),TAT-HA-Apoptin(brown), MPG-Apoptin (light blue) and TAT-E4of4(purple) Finally we have quantitative data. For supporting this results we need qualitative results.When cells going to apoptosis, they change morphology We made use of this data, we decided take a photos of exposured cancer cells. We seeded colon and breast cancer cells on 12 well plate. Cancer cells co-cultured our proteins at desired dosage.after 24 hours, we took photos.

                                          MCF-7 Breast Cancer Cells Morphology Changes

MCF7 cells morphology changes can be visualized below. Photos are taken 24 h after treating with apoptotic proteins and with 20x focus.

  HT-29  Colon Cancer Cells Morphology Changes

HT29 cells morphology changes can be visualized below. Photos are taken 24 h after treating with apoptotic proteins and with 20x focus