Team:ZJU-China/Results
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.
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 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.
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 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.
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.
Figure 4. Western blotting results of immunoprecipitation of scFv-Fc.
Expression of mamC-ZZ
Optimum Concentration of IPTG for 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.
Figure 5. Western blot results of GST mamC-ZZ under different inducing IPTG concentration.
As Figure 5 is shown above, the best concentration for expressing was 2 mM of IPTG.
Optimum Inducion Time for 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.
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.
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.
As for input, GST mamC-ZZ was incubated with purified FLAG scFv-Fc overnight at 4°C (Figure 8B). The left block was incubated with rabbit anti-FLAG (binds to FLAG® tag sequence) antibody and goat anti-rabbit IgG H&L (HRP), and the right block was only incubated with goat anti-rabbit IgG H&L (HRP).
Due to the similar molecular weight of scFv-Fc and mamC-ZZ, we designed thrombin-digestion before western blotting. Thus, scFv-Fc will appear at about 54kDa, digested mamC-ZZ will appear at about 26kDa by interacting with the secondary antibody directly. All the samples were digested by thrombin to cut off any potential GST region.
The mixture of GST mamC-ZZ and FLAG scFv-Fc was incubated with anti-FLAG resin (GenScript, Nanjing, CN) for 1 h. Figure 8A indicated the FLAG scFv-Fc and the interacted mamC-ZZ
was then immobilized on the resin, whereas the unbound proteins were washed away with TBS. Subsequently, the protein–protein complex was eluted. The right block was only
incubated with the secondary antibody, improving mamC-ZZ was pulled down by the interaction of scFv-Fc.
Figure 8. Western blot results of mamC-ZZ and scFv which introduce different primary antibody. (A) One block was incubated with rabbit anti-DDDDK tag 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 show the specific position of mamC-ZZ. (B) Western blot results of input control block.
Furthermore, according to lane 1 and lane 2 (Figure 8A), purified proteins showed stronger interaction than unpurified proteins. However, it suggested an 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.
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.
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 scFv-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).
Culture of Magnetotactic Bacteria
Microaerobic Culture
As magnetotactic bacteria needed microaerobic conditions to grow, we chose liquid medium rather than solid for the growth of bacteria. In order to show the change of oxygen content in the culture medium visibly, we added resazurin into the medium. Resazurin is an indicator of dissolved oxygen. It turns blue when there is dissolved oxygen in the solution and turns red when there is no oxygen. The results showed that the color of the liquid medium which were added into MSR-1 were obviously different from that without the addition of MSR-1 (Figure 11), indicating that the dissolved oxygen may be consumed out by MSR-1.
Figure 11. Liquid medium cultured MSR-1 (red test tubes) contrast to the blank control (blue test tubes).
Bacterial PCR of MSR-1
To further demonstrate that the magnetotactic bacteria was truly grown in the liquid medium, bacterial PCR was carried out using specific primers designed from the 16srDNA of M. gryphiswaldense and used E.coli and Agrobacterium as the negative control. As the results of bacterial PCR showed, there was a bright band in lane2, while no bands in lane 3 and lane 4, demonstrating that MSR-1 was successfully grown up (Figure 12).
Figure 12. Results of the bacterial PCR.
Future Work
Due to the spread of the COVID-19 virus, the majority of our lab work has been delayed. Though we have tried our best to carry out relative experiments to demonstrate our design, there are still many lab work have not been completed. We do believe that the functionalized magnetosomes can make a great impact in the area of MRI contrast agent, and we have listed the future plans for our project to obtain the genetic modification magnetosomes ultimately.
Our future plans are divided into two major parts: construction of functionalized magnetosomes and specificity validation.
Construction of Functionalized Magnetosomes
Acquisition of Genetic Modification Magnetotactic Bacteria
To obtain the modified magnetosomes, we need to obtain the MSR-1 strain which is able to encoding mamC-ZZ protein, and this will be realized by the bi-parental conjugation of the donor (E.coli) harboring recombinant plasmid and the recipient, MSR-1. To increase the amount of ZZ protein on the recombinant magnetosomes, we will use a mamC mutant strain as our recipient, which is constructed by allelic gene replacement by a gentamicin resistance gene[2]. Both the recipient strain and the recombinant strain will be selected by antibiotic resistant culture, respectively.
Bacterial Strains Cultures and the Purification of Magnetosomes
MSR-1 will be cultured in the Na-lactate medium at 30°C for 38–40 h. The contains and concentrates of the medium refers to some recent researchs[3]. Bacteria cells will be collected by centrifugation with PBS (pH 7.4) and then disrupted by two passes through a French Press. Magnetosomes from the disrupted cells will be collected at the bottom of a tube by neodymium-iron-boron magnets that produced an inhomogeneous magnetic field. After removing the supernatant, the collected magnetosomes should be washed eight to ten times with PBS while agitating via low level ultrasonication. Transmission electronic microscopy (TEM) will be used for the examination of purified magnetosomes. Besides that, the TEM image that shows the morphology of magnetosomes can be a preliminary basis for testing the functionalized magnetosomes and the normal. At last, the purified magnetosomes will be lyophilized using a freeze drier for 20 h and will be stored at -20°C as the previous purification and storage protocol guides[4].
Self-assembly of ZZ Modified Magnetosomes and scFv
Based on our experiment results that the mamC-ZZ fusion protein and scFv-Fc fusion protein can bind together strongly and specifically in vitro, we will mix magnetosomes and the purified scFv-Fc proteins with different molar ratios under the same conditions as the previous experiments and use TEM and Zeta Potential Analyzer to detect the morphology of the magnetosome-scFv complexes and screen out the optimal ratio at which the magnetosomes can bind the most scFv on their surfaces.
Increase the Production of Magnetosomes
It is necessary that the yield of magnetosomes should be high enough for commercial applications in the future. At the genetic level, fine tuning such as enhance the expression of an ATPase gene around the putative elongation factor-G gene of the bacterial chromosome of MSR-1 can increase the production of magnetosomes[5]. In terms of mass cultivation, we have protocols for different scales of fermenters. The major restricting factor of the production of magnetosomes is the concentration of dissolved oxygen in the medium. Through changing the string speed by detecting the concentration of magnetotactic bacteria to regulated the concentration of dissolved oxygen timely and using chemostat culture technique, the highest yield could reach 88.59 mg/day, theoretically[6].
Cytotoxicity and Immune Responses Assay
Although magnetosomes are biocompatible, there are several reports say that magnetosomes are toxic to mosquitos and plasmodium[7], which means that they may also be toxic to human bodies. So, for the safety and thorough consideration, cytotoxicity and immune responses assay will be carried out by MTT assay in mouse cell lines such as BHK-21 cells. Cells will be seeded in 96-well plates and incubated for 24h after the addition of magnetosomes, and then accessed by the MTT colorimetric reaction. Absorbance of color should be measured at 485nm by a plate reader and the percent of cell viability should be calculated according to the following equation: Cell viability (%) = [OD490(Sample)/OD(490control)]*100. Besides, as a bacterial product, the entry of magnetosomes into human bodies may causes immune responses, so we will carry out MTT assay for T cell proliferation and ELISA for detecting total IgG antibodies to determine the immune responses of in vivo experiments. MTT assay for T cell proliferation is similarly operating as it is used for cytotoxicity testing, while ELISA can be simply operated using commercially available kits[4].
Specificity validation
In vitro and in vivo Fluorescence Imaging Assays of Magnetosome-scFv Complexes
For the validation of targeting ability and specificity of our ZZ-modified magnetosomes in vitro and in vivo, we will use confocal-laser scanning microscope or flow cytometry to analyze the fluorescence. We will use MDA-MB-468 cells and 293 A cells to carry out contrast experiments. All sample cells and control cells will be seeded at a same density into confocal dishes and cultured for a same period for the following imaging experiments. Though we have not obtained the magnetosomes, yet we are carrying out this part using mamC-ZZ and scFv-Fc complexes as a replacement of magnetosomes to validate its specificity. Sadly, we may not finish this experiment by the wiki freeze, this validation has to be written here.
For in vivo fluorescence imaging, HER2 tumor-baring nude mice will be obtained by injecting PBS containing about 1×107 tumor cells per 100 c into the right flank of the 6-7-week-old female BALB/c nude mice. In vivo fluorescence imaging will be performed 2 weeks after tumor implantation. 1 mg magnetosome-scFv complexes in 200 μL PBS are planned to be intravenously administered into the tumor-bearing nude mice via tail vein. And the fluorescence images of the mice and their main organs will be taken half an hour after the injection.
In vivo MR Imaging Study
To test the in vivo targeting ability of our magnetosome-scFv complexes, MR imaging will be performed on a 3.0T MR imaging system at the first affiliated hospital of Zhejiang University School of Medicine. The MR imaging study will be carried out on the same tumor-bearing nude mice for the in vivo fluorescence assay, and will be conducted by T2-weighted MR imaging with the strength field of 3.0T at different time points after the complexes administered via tail vein.
Summary
All the experiment above are designed with detailed consideration and with the reference to large relative research. And we have all the instruments and proven protocols to carry out our future experiments, especially the in vivo experiments. Thankfully, Prof. Feng Chen from the Department of Radiology, the First Affiliated Hospital of Zhejiang University School of Medicine, provide us with the opportunity to use the MR imagine system and the nude mice kindly, so that we can carry out the in vivo experiments safely and scientifically. We do believe that our functionalized magnetosomes can make a great progress on the MRI detection of breast cancer and provide a new technology on specific imaging.
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.
[2]. Xu J, Hu J, Liu L, Li L, Wang X, Zhang H, Jiang W, Tian J, Li Y, Li J. Surface expression of protein A on magnetosomes and capture of pathogenic bacteria by magnetosome/antibody complexes. Front Microbiol. 2014 Apr 3;5:136.
[3]. Yang J, Li S, Huang X, Tang T, Jiang W, Zhang T, Li Y. A key time point for cell growth and magnetosome synthesis of Magnetospirillum gryphiswaldense based on real-time analysis of physiological factors. Front Microbiol. 2013 Jul 24;4:210.
[4]. Xiang L, Wei J, Jianbo S, Guili W, Feng G, Ying L. Purified and sterilized magnetosomes from Magnetospirillum gryphiswaldense MSR-1 were not toxic to mouse fibroblasts in vitro. Lett Appl Microbiol. 2007 Jul;45(1):75-81
[5]. Liu J, Ding Y, Jiang W, Tian J, Li Y, Li J. A mutation upstream of an ATPase gene significantly increases magnetosome production in Magnetospirillum gryphiswaldense. Appl Microbiol Biotechnol. 2008 Dec;81(3):551-8.
[6]. Liu Y, Li GR, Guo FF, Jiang W, Li Y, Li LJ. Large-scale production of magnetosomes by chemostat culture of Magnetospirillum gryphiswaldense at high cell density. Microb Cell Fact. 2010 Dec 12;9:99.
[7]. Murugan K, Wei J, Alsalhi MS, Nicoletti M, Paulpandi M, Samidoss CM, Dinesh D, Chandramohan B, Paneerselvam C, Subramaniam J, Vadivalagan C, Wei H, Amuthavalli P, Jaganathan A, Devanesan S, Higuchi A, Kumar S, Aziz AT, Nataraj D, Vaseeharan B, Canale A, Benelli G. Magnetic nanoparticles are highly toxic to chloroquine-resistant Plasmodium falciparum, dengue virus (DEN-2), and their mosquito vectors. Parasitol Res. 2017 Feb;116(2):495-502.
[8]. Xiang Z, Yang X, Xu J, Lai W, Wang Z, Hu Z, Tian J, Geng L, Fang Q. Tumor detection using magnetosome nanoparticles functionalized with a newly screened EGFR/HER2 targeting peptide. Biomaterials. 2017 Jan;115:53-64.
