Line 298: | Line 298: | ||
<p> | <p> | ||
− | If not specified, all expression chassis of scFv | + | If not specified, all expression chassis of scFv is SHuffle<sup>®</sup>. The supernatant and the pellet of cell lysate was analyzed by SDS–PAGE, corresponding to lane 1 and |
− | lane 2, respectively. According to the Coomassie staining result below, we pointed that scFv-Fc was presented in the correct weight and expression level was higher in | + | lane 2, respectively. According to the Coomassie staining result below, we pointed out that scFv-Fc was presented in the correct weight and expression level was higher in |
supernatant rather than pellet, indicating the solubility of this fusion protein. | supernatant rather than pellet, indicating the solubility of this fusion protein. | ||
</p> | </p> | ||
Line 306: | Line 306: | ||
<div class="imgbox"> | <div class="imgbox"> | ||
<img src="https://static.igem.org/mediawiki/2020/9/9a/T--ZJU-China--Results_fig1.jpg"></img> | <img src="https://static.igem.org/mediawiki/2020/9/9a/T--ZJU-China--Results_fig1.jpg"></img> | ||
− | <h6> | + | <h6>Figure 1. Coomassie staining results of scFv-Fc to investigate the solubility.</h6> |
</div> | </div> | ||
<br> | <br> | ||
Line 315: | Line 315: | ||
− | <h2>Optimum concentration of IPTG for | + | <h2>Optimum concentration of IPTG for scFv-Fc expression</h2> |
<p> | <p> | ||
We carried out a gradient experiment to investigate the optimum concentration of IPTG for expressing. The transformed cells were grown until OD600 reached 0.6, we introduced | We carried out a gradient experiment to investigate the optimum concentration of IPTG for expressing. The transformed cells were grown until OD600 reached 0.6, we introduced | ||
− | the expression with IPTG, to a final concentration of 0.5, 1 and 2 mM, respectively. For negative control, IPTG was not introduced. | + | the expression with IPTG, to a final concentration of 0.5, 1, and 2 mM, respectively. For negative control, IPTG was not introduced. |
</p> | </p> | ||
<br> | <br> | ||
Line 324: | Line 324: | ||
<div class="imgbox"> | <div class="imgbox"> | ||
<img src="https://static.igem.org/mediawiki/2020/5/54/T--ZJU-China--Results_fig2.jpg" alt=""> | <img src="https://static.igem.org/mediawiki/2020/5/54/T--ZJU-China--Results_fig2.jpg" alt=""> | ||
− | <h6> | + | <h6>Figure 2. Western blotting results of scFv-Fc under different inducing concentration of IPTG.</h6> |
</div> | </div> | ||
<br> | <br> | ||
<p> | <p> | ||
− | As | + | As Figure 2 shows above, the best concentration for expressing was 2 mM of IPTG. There was a small amount of expression without IPTG induction, probably due to promoter leakage. |
The expression level of IPTG decreased first and then increased with the increase of IPTG concentration. This may cause by the inhibitory effect of IPTG on bacterial growth. | The expression level of IPTG decreased first and then increased with the increase of IPTG concentration. This may cause by the inhibitory effect of IPTG on bacterial growth. | ||
</p> | </p> | ||
− | <h2>Optimum induction time for | + | <h2>Optimum induction time for scFv-Fc expression</h2> |
<p> | <p> | ||
− | A gradient experiment was performed to make sure that the target protein was induced and to figure out the best time for expressing. When OD600 reached 0.6, | + | A gradient experiment was performed to make sure that the target protein was induced and to figure out the best time for expressing. When OD600 reached 0.6, 2 mM of IPTG was |
− | induced. The expression time was 0, 1, 8 and | + | induced. The expression time was 0, 1, 8, and 24 h, respectively. For negative control, none inducer was introduced. |
</p> | </p> | ||
<br> | <br> | ||
Line 342: | Line 342: | ||
<div class="imgbox"> | <div class="imgbox"> | ||
<img src="https://static.igem.org/mediawiki/2020/b/bf/T--ZJU-China--Results_fig3.jpg" alt=""> | <img src="https://static.igem.org/mediawiki/2020/b/bf/T--ZJU-China--Results_fig3.jpg" alt=""> | ||
− | <h6> | + | <h6>Figure 3. Western blotting results of scFv-Fc under different induction time.</h6> |
</div> | </div> | ||
<br> | <br> | ||
<p> | <p> | ||
− | As | + | As Figure 3 shows above, the longer time for expressing, the higher expression level the cells reached. In the next work, the expression period was carried out under 2 mM of IPTG |
inducing and 24 h of additional incubating. | inducing and 24 h of additional incubating. | ||
</p> | </p> | ||
Line 354: | Line 354: | ||
<p> | <p> | ||
We perform the immunoprecipitation (IP) to obtain purified FLAG-tagged protein. IP has been described in detail in the experiment section. The target protein appeared at 54kDa. | We perform the immunoprecipitation (IP) to obtain purified FLAG-tagged protein. IP has been described in detail in the experiment section. The target protein appeared at 54kDa. | ||
− | Nevertheless, there were a lot of proteins in the high molecular weight part, because scFv would form polymer, which was immobilized as well. At the same time, there was | + | Nevertheless, there were a lot of proteins in the high molecular weight part, because scFv would form a polymer, which was immobilized as well. At the same time, there was |
protein in small the molecular weight part, which may be caused by the protein truncating easily at the junction. | protein in small the molecular weight part, which may be caused by the protein truncating easily at the junction. | ||
</p> | </p> | ||
<div class="imgbox"> | <div class="imgbox"> | ||
<img src="https://static.igem.org/mediawiki/2020/6/68/T--ZJU-China--Results_fig4.jpg" alt=""> | <img src="https://static.igem.org/mediawiki/2020/6/68/T--ZJU-China--Results_fig4.jpg" alt=""> | ||
− | <h6> | + | <h6>Figure 4. Western blotting results of immunoprecipitation of scFv-Fc.</h6> |
</div> | </div> | ||
</div> | </div> | ||
Line 371: | Line 371: | ||
<br> | <br> | ||
<br> | <br> | ||
− | <h2>Optimum | + | <h2>Optimum concentration of IPTG for mamC-ZZ expression</h2> |
<p> | <p> | ||
A gradient experiment was conducted to investigate the optimum IPTG concentration for GST mamC-ZZ expression. The transformed bacteria were cultured until OD600 reached 0.6, we | A gradient experiment was conducted to investigate the optimum IPTG concentration for GST mamC-ZZ expression. The transformed bacteria were cultured until OD600 reached 0.6, we | ||
− | induced the expression with IPTG, to a final concentration of 1, 2 and 3mM, respectively. For negative control, IPTG was not introduced. | + | induced the expression with IPTG, to a final concentration of 1, 2, and 3mM, respectively. For negative control, IPTG was not introduced. |
</p> | </p> | ||
<div class="imgbox"> | <div class="imgbox"> | ||
<img src="https://static.igem.org/mediawiki/2020/thumb/e/eb/T--ZJU-China--Results_fig5.jpg/800px-T--ZJU-China--Results_fig5.jpg" alt=""> | <img src="https://static.igem.org/mediawiki/2020/thumb/e/eb/T--ZJU-China--Results_fig5.jpg/800px-T--ZJU-China--Results_fig5.jpg" alt=""> | ||
− | <h6>Fig5. Western | + | <h6>Fig5. Western blot results of GST mamC-ZZ under different inducing IPTG concentration. As results shown above, the best concentration for expressing was 2 mM of IPTG.</h6> |
</div> | </div> | ||
<br> | <br> | ||
− | <p>As Fig5 | + | <p>As Fig5 is shown above, the best concentration for expressing was 2 mM of IPTG. </p> |
− | <h2>Optimum | + | <h2>Optimum inducion time for mamC-ZZ expression</h2> |
<p>A gradient experiment was carried out to investigate the optimum inducing time for GST mamC-ZZ expression. The transformed bacteria were cultured until OD600 reached 0.6 and | <p>A gradient experiment was carried out to investigate the optimum inducing time for GST mamC-ZZ expression. The transformed bacteria were cultured until OD600 reached 0.6 and | ||
− | induced the expression with IPTG. In a single experimental blocks, a group of gradient expressing time, which set from | + | induced the expression with IPTG. In a single experimental blocks, a group of gradient expressing time, which set from 1 h to 4 h, was tested. |
</p> | </p> | ||
<div class="imgbox"> | <div class="imgbox"> | ||
<img src="https://static.igem.org/mediawiki/2020/thumb/c/cf/T--ZJU-China--Results_fig6.jpg/799px-T--ZJU-China--Results_fig6.jpg" alt=""> | <img src="https://static.igem.org/mediawiki/2020/thumb/c/cf/T--ZJU-China--Results_fig6.jpg/799px-T--ZJU-China--Results_fig6.jpg" alt=""> | ||
− | <h6> | + | <h6>Figure 6. Western blot results of GST mamC-ZZ under different induction time.</h6> |
</div> | </div> | ||
<br> | <br> | ||
Line 397: | Line 397: | ||
<p> | <p> | ||
Immunoprecipitation (IP) was performed to obtain purified GST-tagged protein and investigate the optimum condition of purification. IP has been described in detail in the | Immunoprecipitation (IP) was performed to obtain purified GST-tagged protein and investigate the optimum condition of purification. IP has been described in detail in the | ||
− | experiment section. The target protein appeared at 52kDa. A group of gradient glutathione resin, which added as single,double and triple volume of GST mamC-ZZ solution, was | + | experiment section. The target protein appeared at 52kDa. A group of gradient glutathione resin, which added as single, double and triple volume of GST mamC-ZZ solution, was |
tested. | tested. | ||
</p> | </p> | ||
<div class="imgbox"> | <div class="imgbox"> | ||
<img src="https://static.igem.org/mediawiki/2020/thumb/0/05/T--ZJU-China--Results_fig7.jpg/800px-T--ZJU-China--Results_fig7.jpg" alt=""> | <img src="https://static.igem.org/mediawiki/2020/thumb/0/05/T--ZJU-China--Results_fig7.jpg/800px-T--ZJU-China--Results_fig7.jpg" alt=""> | ||
− | <h6> | + | <h6>Figure 7. Coomassie staining results of GST mamC-ZZ under different purification conditions</h6> |
</div> | </div> | ||
<br> | <br> | ||
<p> | <p> | ||
− | As results shown above, the efficiency of purification did not demonstrate a significant difference among different glutathione resin | + | As results are shown above, the efficiency of purification did not demonstrate a significant difference among different glutathione resin volumes which added in. In the following work, we added equal glutathione resin as GST mamC-ZZ solution to purify the recombinant protein. |
− | + | ||
</p> | </p> | ||
Line 414: | Line 413: | ||
<div class="section services" id="protein_interaction"> | <div class="section services" id="protein_interaction"> | ||
<div class="container1"> | <div class="container1"> | ||
− | <h2 style="line-height:1.5;">Protein | + | <h2 style="line-height:1.5;">Protein Interaction</h2> |
<br> | <br> | ||
<br> | <br> | ||
<h2>Immunoprecipitation</h2> | <h2>Immunoprecipitation</h2> | ||
<p> | <p> | ||
− | To prove that | + | To prove that mamC-ZZ can combine with scFv-Fc, we conducted the immunoprecipitation experiments. |
<br> | <br> | ||
Lane 1 stands for input, which was loaded with cell lysate of which obtained mamC-ZZ and purified. Lane 2 was loaded with IP products of cell lysate of which obtained mamC-ZZ | Lane 1 stands for input, which was loaded with cell lysate of which obtained mamC-ZZ and purified. Lane 2 was loaded with IP products of cell lysate of which obtained mamC-ZZ | ||
Line 428: | Line 427: | ||
<div class="imgbox"> | <div class="imgbox"> | ||
<img src="https://static.igem.org/mediawiki/2020/d/d6/T--ZJU-China--Results_fig8.png" alt=""> | <img src="https://static.igem.org/mediawiki/2020/d/d6/T--ZJU-China--Results_fig8.png" alt=""> | ||
− | <h6> | + | <h6>Figure 8. Western blot results of mamC-ZZ and scFv which introduce different primary antibody. One block was incubated with rabbit anti-DDDDK tag (binds to FLAG® tag sequence) |
− | antibody and goat anti-rabbit IgG H&L (HRP), and another block was only incubated with goat anti-rabbit IgG H&L (HRP), which can interact with mamC-ZZ by Fc region and | + | antibody and goat anti-rabbit IgG H&L (HRP), and another block was only incubated with goat anti-rabbit IgG H&L (HRP), which can interact with mamC-ZZ by Fc region and shows |
− | the specific position of mamC-ZZ; B. Western-blot results of input control block.</h6> | + | the specific position of mamC-ZZ; B. Western-blot results of the input control block.</h6> |
</div> | </div> | ||
<br> | <br> | ||
Line 453: | Line 452: | ||
<div class="imgbox"> | <div class="imgbox"> | ||
<img src="https://static.igem.org/mediawiki/2020/thumb/8/8d/T--ZJU-China--Results_cell_culture1.jpg/800px-T--ZJU-China--Results_cell_culture1.jpg" alt=""> | <img src="https://static.igem.org/mediawiki/2020/thumb/8/8d/T--ZJU-China--Results_cell_culture1.jpg/800px-T--ZJU-China--Results_cell_culture1.jpg" alt=""> | ||
− | <h6> | + | <h6>Figure 9. Images of MDA-MB-253 and MDA-MB-453 at different time after subculture. (A). This image of MDA-MB-231 was taken one day after subculturing, the amount of cell is |
− | low; (B).This image of MDA-MB-231 was taken | + | low; (B).This image of MDA-MB-231 was taken four days after subculturing, tumor cell was at good survival conditions and developed a high cell intensity; (C).This |
− | image of MDA-MB-453 in medium intensity was taken at six days after subculturing; (D).Ten days after subculturing, MDA-MB-453 showed a great cell intensity, and morphology | + | image of MDA-MB-453 in medium intensity was taken at six days after subculturing; (D).Ten days after subculturing, MDA-MB-453 showed a great cell intensity, and the morphology |
of some tumor cells has transformed account for long-time cultivation.</h6> | of some tumor cells has transformed account for long-time cultivation.</h6> | ||
</div> | </div> | ||
<br> | <br> | ||
<p> | <p> | ||
− | As images shown above, both | + | As images are shown above, both MDA-MB-453 and MDA-MB-231 was cultured successfully and prepared to attend further experiments steps. |
</p> | </p> | ||
Line 469: | Line 468: | ||
<div class="imgbox"> | <div class="imgbox"> | ||
<img src="https://static.igem.org/mediawiki/2020/c/ce/T--ZJU-China--Results_fig10_newone.jpg" alt=""> | <img src="https://static.igem.org/mediawiki/2020/c/ce/T--ZJU-China--Results_fig10_newone.jpg" alt=""> | ||
− | <h6> | + | <h6>Figure 10. Flow cytometry results of MDA-MB-453 and MDA-MB-231 after incubated with scFv-Fc.</h6> |
</div> | </div> | ||
<br> | <br> | ||
<br> | <br> | ||
<p> | <p> | ||
− | As results shown above, the fluorescence intensity of MDA-MB-453 cells which incubated with scFv-Fc was significantly higher than the other MDA-MB-453 cells from negative | + | As results are shown above, the fluorescence intensity of MDA-MB-453 cells which incubated with scFv-Fc was significantly higher than the other MDA-MB-453 cells from the negative |
− | control group, which indicated that scFv-Fc could specifically bind with certain target on MDA-MB-453 membrane( | + | control group, which indicated that scFv-Fc could specifically bind with the certain target on MDA-MB-453 membrane (Figure 10A). |
<br> | <br> | ||
− | At the same time, we can also see that MDA-MB-231 has obvious fluorescence peak after incubated by scFv-Fc( | + | At the same time, we can also see that MDA-MB-231 has an obvious fluorescence peak after incubated by scFv-Fc (Figure 10B). |
<br> | <br> | ||
− | Then, the following analysis shows that although scFc-Fc can target both MDA-MB-453 and MDA-MB-231 cells, the fluorescence peaks | + | Then, the following analysis shows that although scFc-Fc can target both MDA-MB-453 and MDA-MB-231 cells, the fluorescence peaks were significantly different. Obviously, the |
high HER2 expression cell line (MDA-MB-453) showed a higher fluorescence than that of the low HER2 expression cell line (MDA-MB-231), indicating that scFv-Fc is more targeted | high HER2 expression cell line (MDA-MB-453) showed a higher fluorescence than that of the low HER2 expression cell line (MDA-MB-231), indicating that scFv-Fc is more targeted | ||
− | to HER2, and can distinguish breast cancer cells with high and low expression of HER2 ( | + | to HER2, and can distinguish breast cancer cells with high and low expression of HER2 (Figure 10C). And the fluorescence of MDA-MB-453 was about 10 times higher than |
− | MDA-MB-231’s, which was corresponding with the difference of HER2 expression level between HER2 positive and negative cells mentioned in existing studies <a href="#reference"><sup>[1]</sup></a>. | + | MDA-MB-231’s, which was corresponding with the difference of HER2 expression level between HER2 positive and negative cells mentioned in existing studies<a href="#reference"><sup>[1]</sup></a>. |
<br> | <br> | ||
To put it more bluntly, compared with MDA-MB-231 which is HER2-negative, MDA-MB-453 had significantly higher fluorescence intensity when both cells were alive. This results | To put it more bluntly, compared with MDA-MB-231 which is HER2-negative, MDA-MB-453 had significantly higher fluorescence intensity when both cells were alive. This results | ||
− | indicates clearly that scFv can specifically bind to HER2 on tumor cell membrane ( | + | indicates clearly that scFv can specifically bind to HER2 on tumor cell membrane (Figure 10D). |
</p> | </p> | ||
</div> | </div> | ||
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<div class="section our-team" id="reference"> | <div class="section our-team" id="reference"> | ||
<div class="container1"> | <div class="container1"> | ||
− | <h2> | + | <h2>References</h2> |
<p> | <p> | ||
[1]. Press, M. F., Pike, M. C., Chazin, V. R., Hung, G., Udove, J. A., Markowicz, M., Danyluk, J., Godolphin, W., Sliwkowski, M., & Akita, R. (1993). Her-2/neu expression in node-negative breast cancer: direct tissue quantitation by computerized image analysis and association of overexpression with increased risk of recurrent disease. <i>Cancer research</i>, 53(20), 4960–4970. | [1]. Press, M. F., Pike, M. C., Chazin, V. R., Hung, G., Udove, J. A., Markowicz, M., Danyluk, J., Godolphin, W., Sliwkowski, M., & Akita, R. (1993). Her-2/neu expression in node-negative breast cancer: direct tissue quantitation by computerized image analysis and association of overexpression with increased risk of recurrent disease. <i>Cancer research</i>, 53(20), 4960–4970. | ||
</p> | </p> | ||
+ | <br> | ||
+ | <br> | ||
+ | <br> | ||
<br> | <br> | ||
<br> | <br> |
Revision as of 04:21, 27 October 2020
Results
Expression of scFv-Fc
If not specified, all expression chassis of scFv is SHuffle®. The supernatant and the pellet of cell lysate was analyzed by SDS–PAGE, corresponding to lane 1 and lane 2, respectively. According to the Coomassie staining result below, we pointed out that scFv-Fc was presented in the correct weight and expression level was higher in supernatant rather than pellet, indicating the solubility of this fusion protein.
![](https://static.igem.org/mediawiki/2020/9/9a/T--ZJU-China--Results_fig1.jpg)
Figure 1. Coomassie staining results of scFv-Fc to investigate the solubility.
Besides, scFv-Fc was also expressed in BL21 (DE3), which meant the absence of two disulfide bonds, leading to the incorrect folding of scFv. Therefore, the protein expressed in BL21 (DE3) cannot interact with HER2 theoretically.
Optimum concentration of IPTG for scFv-Fc expression
We carried out a gradient experiment to investigate the optimum concentration of IPTG for expressing. The transformed cells were grown until OD600 reached 0.6, we introduced the expression with IPTG, to a final concentration of 0.5, 1, and 2 mM, respectively. For negative control, IPTG was not introduced.
![](https://static.igem.org/mediawiki/2020/5/54/T--ZJU-China--Results_fig2.jpg)
Figure 2. Western blotting results of scFv-Fc under different inducing concentration of IPTG.
As Figure 2 shows above, the best concentration for expressing was 2 mM of IPTG. There was a small amount of expression without IPTG induction, probably due to promoter leakage. The expression level of IPTG decreased first and then increased with the increase of IPTG concentration. This may cause by the inhibitory effect of IPTG on bacterial growth.
Optimum induction time for scFv-Fc expression
A gradient experiment was performed to make sure that the target protein was induced and to figure out the best time for expressing. When OD600 reached 0.6, 2 mM of IPTG was induced. The expression time was 0, 1, 8, and 24 h, respectively. For negative control, none inducer was introduced.
![](https://static.igem.org/mediawiki/2020/b/bf/T--ZJU-China--Results_fig3.jpg)
Figure 3. Western blotting results of scFv-Fc under different induction time.
As Figure 3 shows above, the longer time for expressing, the higher expression level the cells reached. In the next work, the expression period was carried out under 2 mM of IPTG inducing and 24 h of additional incubating.
Purification of the proteins
We perform the immunoprecipitation (IP) to obtain purified FLAG-tagged protein. IP has been described in detail in the experiment section. The target protein appeared at 54kDa. Nevertheless, there were a lot of proteins in the high molecular weight part, because scFv would form a polymer, which was immobilized as well. At the same time, there was protein in small the molecular weight part, which may be caused by the protein truncating easily at the junction.
![](https://static.igem.org/mediawiki/2020/6/68/T--ZJU-China--Results_fig4.jpg)
Figure 4. Western blotting results of immunoprecipitation of scFv-Fc.
Expression of mamC-ZZ
Optimum concentration of IPTG for mamC-ZZ expression
A gradient experiment was conducted to investigate the optimum IPTG concentration for GST mamC-ZZ expression. The transformed bacteria were cultured until OD600 reached 0.6, we induced the expression with IPTG, to a final concentration of 1, 2, and 3mM, respectively. For negative control, IPTG was not introduced.
![](https://static.igem.org/mediawiki/2020/thumb/e/eb/T--ZJU-China--Results_fig5.jpg/800px-T--ZJU-China--Results_fig5.jpg)
Fig5. Western blot results of GST mamC-ZZ under different inducing IPTG concentration. As results shown above, the best concentration for expressing was 2 mM of IPTG.
As Fig5 is shown above, the best concentration for expressing was 2 mM of IPTG.
Optimum inducion time for mamC-ZZ expression
A gradient experiment was carried out to investigate the optimum inducing time for GST mamC-ZZ expression. The transformed bacteria were cultured until OD600 reached 0.6 and induced the expression with IPTG. In a single experimental blocks, a group of gradient expressing time, which set from 1 h to 4 h, was tested.
![](https://static.igem.org/mediawiki/2020/thumb/c/cf/T--ZJU-China--Results_fig6.jpg/799px-T--ZJU-China--Results_fig6.jpg)
Figure 6. Western blot results of GST mamC-ZZ under different induction time.
Purification of the proteins
Immunoprecipitation (IP) was performed to obtain purified GST-tagged protein and investigate the optimum condition of purification. IP has been described in detail in the experiment section. The target protein appeared at 52kDa. A group of gradient glutathione resin, which added as single, double and triple volume of GST mamC-ZZ solution, was tested.
![](https://static.igem.org/mediawiki/2020/thumb/0/05/T--ZJU-China--Results_fig7.jpg/800px-T--ZJU-China--Results_fig7.jpg)
Figure 7. Coomassie staining results of GST mamC-ZZ under different purification conditions
As results are shown above, the efficiency of purification did not demonstrate a significant difference among different glutathione resin volumes which added in. In the following work, we added equal glutathione resin as GST mamC-ZZ solution to purify the recombinant protein.
Protein Interaction
Immunoprecipitation
To prove that mamC-ZZ can combine with scFv-Fc, we conducted the immunoprecipitation experiments.
Lane 1 stands for input, which was loaded with cell lysate of which obtained mamC-ZZ and purified. Lane 2 was loaded with IP products of cell lysate of which obtained mamC-ZZ
and purified. Lane 3 was loaded with IP products of purified mamC-ZZ and scFv. Lastly, lane 4 was loaded only with scFv. All the samples were digested by thrombin to cut off
any potential GST region.
![](https://static.igem.org/mediawiki/2020/d/d6/T--ZJU-China--Results_fig8.png)
Figure 8. Western blot results of mamC-ZZ and scFv which introduce different primary antibody. One block was incubated with rabbit anti-DDDDK tag (binds to FLAG® tag sequence) antibody and goat anti-rabbit IgG H&L (HRP), and another block was only incubated with goat anti-rabbit IgG H&L (HRP), which can interact with mamC-ZZ by Fc region and shows the specific position of mamC-ZZ; B. Western-blot results of the input control block.
The mamC-ZZ could bind secondary antibody by Fc region directly and the band would appear at 26 kDa approximately. Lane 2 indicated that scFv-Fc was immobilized on the resin, meanwhile, the ZZ region specifically interacted with the Fc region and also showed a band (Fig 8. A). Furthermore, lane 3 suggested a more inspiring result, which meant scFv-Fc could bind mamC-ZZ in a complicated environment. These results implied us an easier way to purified and enriched mamC-ZZ from cell lysate directly.
Specifically target HER2
Cultivation of tumor cells
We cultured two kinds of tumor cells which is MDA-MB-253 represented HER2 positive breast cancer and MDA-MB-231 represented HER2 negative breast cancer.
![](https://static.igem.org/mediawiki/2020/thumb/8/8d/T--ZJU-China--Results_cell_culture1.jpg/800px-T--ZJU-China--Results_cell_culture1.jpg)
Figure 9. Images of MDA-MB-253 and MDA-MB-453 at different time after subculture. (A). This image of MDA-MB-231 was taken one day after subculturing, the amount of cell is low; (B).This image of MDA-MB-231 was taken four days after subculturing, tumor cell was at good survival conditions and developed a high cell intensity; (C).This image of MDA-MB-453 in medium intensity was taken at six days after subculturing; (D).Ten days after subculturing, MDA-MB-453 showed a great cell intensity, and the morphology of some tumor cells has transformed account for long-time cultivation.
As images are shown above, both MDA-MB-453 and MDA-MB-231 was cultured successfully and prepared to attend further experiments steps.
Flow Cytometry
MDA-MB-453 and MDA-MB-231 was both incubated with scFv-Fc on ice for 30 minutes, and stained with Fixable Viability Dye eFluor 450 and Alexa Fluor 488 for flow cytometry.
![](https://static.igem.org/mediawiki/2020/c/ce/T--ZJU-China--Results_fig10_newone.jpg)
Figure 10. Flow cytometry results of MDA-MB-453 and MDA-MB-231 after incubated with scFv-Fc.
As results are shown above, the fluorescence intensity of MDA-MB-453 cells which incubated with scFv-Fc was significantly higher than the other MDA-MB-453 cells from the negative
control group, which indicated that scFv-Fc could specifically bind with the certain target on MDA-MB-453 membrane (Figure 10A).
At the same time, we can also see that MDA-MB-231 has an obvious fluorescence peak after incubated by scFv-Fc (Figure 10B).
Then, the following analysis shows that although scFc-Fc can target both MDA-MB-453 and MDA-MB-231 cells, the fluorescence peaks were significantly different. Obviously, the
high HER2 expression cell line (MDA-MB-453) showed a higher fluorescence than that of the low HER2 expression cell line (MDA-MB-231), indicating that scFv-Fc is more targeted
to HER2, and can distinguish breast cancer cells with high and low expression of HER2 (Figure 10C). And the fluorescence of MDA-MB-453 was about 10 times higher than
MDA-MB-231’s, which was corresponding with the difference of HER2 expression level between HER2 positive and negative cells mentioned in existing studies[1].
To put it more bluntly, compared with MDA-MB-231 which is HER2-negative, MDA-MB-453 had significantly higher fluorescence intensity when both cells were alive. This results
indicates clearly that scFv can specifically bind to HER2 on tumor cell membrane (Figure 10D).
References
[1]. Press, M. F., Pike, M. C., Chazin, V. R., Hung, G., Udove, J. A., Markowicz, M., Danyluk, J., Godolphin, W., Sliwkowski, M., & Akita, R. (1993). Her-2/neu expression in node-negative breast cancer: direct tissue quantitation by computerized image analysis and association of overexpression with increased risk of recurrent disease. Cancer research, 53(20), 4960–4970.