We aimed at introducing the Gox1 protein for TNBC treatment which will digest glucose in order to cut the nutrition income of TNBC so as to make ferroptosis happen. In our project, we constructed the plasmid pET-28a-Gox1and pCOLA-CI857-SRRz-GOX1, obtained the protein upon inducing expression. Following that we used glucose oxidase activity detection assay, reduced glutathione (GSH) content assay and cell counting kit-8 (CCK-8) with 4T1 cell lines for protein activity evaluation.

I. Construction recombinant plasmids

Figure 1.1 Construction of pET28a-Gox1 plasmid. Fig.1.1A shows the gel electrophoresis result of PCR for Gox1; Fig.1.1B represents a double enzyme gel cleavage diagram. Fig.1.1C is PCR validation gel map. Fig.1D is monoclone plate.

The target gene Gox1 was synthesized by the company Genscript and the vector pET28a we used is provided by SubCat. Firstly we amplified Gox1 and pET28a through PCR and then plasmid pET28a-Gox1 was constructed by T4 ligation.

From figure 1.1A, we can see that the size of the target gene Gox1 is nearly 1800bp, and it shown as left four highlighting tape which means that we obtained the amplified right target gene fragments.

From figure 1.1B, compared to the control (line2), its plasmid was successfully digested. If the plasmid is cut and linear, it’ll run slower than if it’s not cut. Because the bright band ended up in the right place, it proves that the final enzymatic digestion experiment was successful.

By picking several monoclones (Fig.1.1D), we ran PCR and electrophoresis gel to validate the plasmid construction and E. coli transformation. Fig.1.1C (Gox1, 1800bp around) indicated that we constructed the correct plasmid.

Figure 1.2 electrophoresisgel results of pCOLA-CI857-SRRz-Gox1 plasmid construction

Fig.1.2A: gel map of the vector after Gibson assembly

Fig.1.2B: gel map of the Gox1 gene fragment

Fig.1.2C: PCR identification gel map of PaCYC-QS quorum sensing vector by colony PCR.

Fig.1.2D and 2E: the PCR identification gel map of pCOLA-CI857-SRRz-Gox1 vector by colony PCR.

Figure 1.2 shows the construction results of pCOLA-CI857-SRRz-Gox1 plasmid and its quorum sensing vector PaCYC-QS. Figure 1.2A shows the pCOLA-CI857-SRRz-Gox1 vector constructed after Gibson assembly. Figure 1.2B shows the gel map of the Gox1 gene which encodes Gox1 cassette.

After assembly, we transformed the plasmids into DH10β competent cells (PaCYC-QS vector has been synthesized by the company). After the transformation and cultivation, we conducted colony PCR identification.

Figure 1.2C is the colony PCR identification result of the quorum sensing vector PaCYC-QS. Figure 1.2D is colony PCR identification of the Gox1 gene in the pCOLA-CI857-SRRz-Gox1 plasmid.

Following the positive result of validation, we then transform the plasmids into the BL21(DE3) E. coli for protein expression. Figure 1.2E is the colony PCR identification of the pCOLA-CI857-SRRz-Gox1 plasmid which confirmed that we have obtained the engineered BL21(DE3) E. coli.

II. Plasmid Sequencing and GOX1 protein expression

Figure 2.1. Gene sequencing results and protein expression of pET28a-Gox1 plasmid. Fig.2.1A shows the result of the gene sequencing. Fig.2.1B shows the target protein Gox1 at 65 kDa.

After the PCR validation for the plasmid pET28a-Gox1, we also sent them to the company for sequencing. From Fig.2.1A, the red arrow shows that the sequencing results match correctly. Together with Fig.1C, they prove that the plasmid was successfully constructed and successfully transferred into Escherichia coli DH5α and BL21 through heat shock, respectively.

Subsequently, the IPTG induction expression and protein purification were conducted with pET28a-Gox1 E. coli BL21. As shown in Fig.2.1B, SDS-PAGE gel result indicates that the molecular weight of the target protein GOX1 is 65kDa.

Figure 2.2 Lysis result of heat-induced autolysis plasmid (pCOLA-CI857-SRRz-GOX1 and PaCYC-QS). Figures 2.2A and 2.2B are two replicates.

After overnight incubation of the pCOLA-CI857-SRRz-Gox1 BL21 E. coli, lysis was measured by the microplate reader and monitored to ensure enough lysis. The results in Figure 2.2 prove that the cells were fully lysed, and the protein coded by Gox1 gene will be purified following.

Figure 2.3. SDS-PAGE of pCOLA-CI857-SRRz-Gox1 plasmid protein expression after heat-induced autolysis.

After purifying samples from the lysis of pCOLA-CI857-SRRz-Gox1 BL21 E. coli, we performed SDS-PAGE to verify the expression of the target protein. The size of the target protein, Gox1 is 65kDa, as shown in Figure 2.3, the same of that as Figure 2.1.

III. GOX1 Glucose Oxidase Activity and CCK-8 Test

Figure 3.1 GOX1 protein glucose oxidase activity and CCK-8 Test with 4T1 cells. Fig.3.1A is 4T1 cells (mouse breast cancer cells). Fig.3.1B is glucose oxidase activity values of different concentrations of the protein. Fig.3.1C CCK-8 samples on a 96 well plate. Fig.3.1D CCK-8 activity values.

After the target protein GOX1 is successfully expressed, its function is verified through a series of experiments in order to measure its glucose oxidase activity and inhibition ability against 4T1 cells.

From Fig.3.1A and 3.1B which present the glucose oxidase activity of the protein GOX1. As the protein concentration increases, the glucose oxidase activity also increases. This trend confirmed the positive glucose oxidase activity of protein GOX1 and we can infer that the higher the glucose oxidase activity the easier the protein promotes iron death.

CCK-8 kit provides a rapid and highly sensitive method for measuring cell proliferation and cytotoxicity. The kit uses WST-8 compound that can be reduced by dehydrogenases in mitochondria to form water soluble formazan in orange color. The more the cells, the darker the color; the higher the cytotoxicity, the lighter the color. For the same cells, there is a linear relationship between color intensity and cell numbers which can be easily measured by microplate reader. Herein this assay was used to evaluate the inhibition ability of the protein GOX1 on 4T1 cells which is tumor at an animal stage IV human breast cancer.

In Fig.3.1C which present the result of CCK-8, we can observe that the area of light pink colour with higher concentrations of GOX1 proteins means fewer 4T1cells. After measuring the activity by a microplate reader, we can see that there is a declining trend of 4T1 cells against GOX1 protein concentrations in Fig.3.1D. Therefore, we can conclude that GOX1 proteins possess the inhibition ability against 4T1 cells and higher concentrations of the protein, better suppressed the 4T1 cells under given setting.

Figure 3.2 glucose oxidase activity and CCK-8 activity of the GOX1 protein purified after heat-induced autolysis

Figure 3.2A glucose oxidase activity of the autolysis experimental group.

Figure 3.2B relative CCK-8 activity of the autolysis experimental group.

For the GOX1 protein purified after heat-induced autolysis, we also conducted glucose oxidase activity assay and CCK-8 test. Figure 3.2A shows that when the protein concentration increases, the glucose concentration increases in a positive trend, confirming that the protein is able to promote ferroptosis by consuming glutathione. The CCK-8 test in Figure 3.2B also backs up this inference.

IV. GOX1 GSH Activity Test

Figure 4.1 Gox1 GSH Activity test on 4T1 cells. Fig.4.1A shows a 96 well plate diagram of the GSH standard samples. Fig.4.1B is the GSH activity standard curve. Fig.4.1C shows the 96 well plate diagram of GSH experimental samples. Fig.4.1D shows the relative GSH activity values of experimental samples.

Generally, cells would consume glutathione to convert cytotoxic lipid peroxides to non-toxic lipid alcohols so as to inhibit ferroptosis. Thus we introduced Gox1 to digest glucose, which cuts the nutrition income of TNBC cells which will result in ferroptosis. To confirm our theory, we used the Reduced Glutathione (GSH) Content Assay kit to help measure the glutathione content changes so as to evaluate the protein ability on glutathione digestion. Similar to the CCK-8 assay, the lighter the colour, the lower the glutathione contents.

Fig.4A and Fig.4B is a standard curve of glutathione content served as a reference. By detecting the same sample as CCK-8, in Fig.4C we could draw the similar conclusion that higher concentrations of the protein Gox1 possessed higher glutathione digestion ability.

After being measured by a microplate reader, Fig.4D shows that 64 μg/mL of Gox1 protein and RSL (1 μM) can achieve about the same level of cancer inhibition which could possess a higher potential to promote iron death of TNBC cells.

Figure 4.2 GSH Activity test on 4T1 cells of the GOX1 protein purified after heat-induced autolysis

Figure 4.2A GSH activity standard curve of the autolysis experimental group

Figure 4.2B relative GSH activity of the autolysis experimental group.

GSH Activity Assay was also conducted with the GOX1 protein purified after heat-induced autolysis. In Figure 4.2, compared with the control group, as the protein concentration increases, the GSH content decreases. In other words, higher protein concentration possesses higher glutathione digestion performance, which also means a better effect on promoting 4T1 cell ferroptosis at the given range.

Since our product is purified protein for TNBC targeted treatment, there are still many challenges to implementing this new therapy, including how to administer our protein into the cancer cells for functioning and how to evaluate the drug effect. Even though there is a long go, we will do more research and incorporate more new ideas to develop this TNBC therapy.