Part:BBa_K3111201
mScarlet_DARPin929_StrepII
This part encodes mScarlet_DARPin929. The use of this part enabled us to study the binding and uptake of DARPin929 by conducting cell culture studies on SK-BR-3 HER2+ cells. The DARPin is flanked by red fluorescent protein mScarlet on the N-terminus which allowed visualisation of binding under confocal microscopy and a StrepII-tag downstream of this fusion, which allows for column-based purification of the expressed protein. It is expressed under a T7 promoter and a strong RBS.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 791
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 652
- 1000COMPATIBLE WITH RFC[1000]
Experimental Results
DNA Analysis and Cloning
We started by cloning BBa_K3111201 into pSB1C3 vector. In order to investigate whether the ligation was successful, we picked 4 colonies from the plate containing the transformed DΗ5α, grown into 5 mL cultures, miniprepped and conducted a test digest with restriction enzymes BamHI and XbaI.
We expected bands at 2760 bp and 792 bp and those were obtained only for colony B as observed in Figure 1. Thus, the miniprepped plasmid obtained from that colony was then transformed into BL21 (DE3) into order to proceed with 50 mL cultures to express the protein.
Day 1
Different batches of BL21 (DE3) competent cells were transformed with pSB1C3 plasmids containing BBa_K3111201 sequence coding for mScarlet + DARPin fusion protein. Transformed cells were grown in LB agar plates containing chloramphenicol and glucose. Plates were incubated at 37°C overnight.
Day 2
Transformed colonies containing pSB1C3_ BBa_K3111201 were used to prepare overnight starter cultures containing a total of 5 mL LB broth and chloramphenicol (5 μL). Cultures were incubated at 37 °C overnight.
Day 3
A 50 mL scale-up cultures was prepared from a single starter culture containing cells carrying pSB1C3 + BBa_K3111201. The culture was incubated at 37°C until it reached an OD of 0.6. Once they reached OD 0.6, the cultures were induced by addition of 400 μΜ IPTG. The cultures were left to grow again overnight at 37 °C.
Day 4
The culture was collected and transferred into a 50 mL falcon tube. It was spun for 10 minutes at 5000 rpm in order to pellet the cells. Then the supernatant was discarded and the pellet frozen at -80 °C.
Protein analysis
In order to observe whether the mScarlet_DARPin929 fusion protein was successfully expressed we analysed our cell pellet using SDS PAGE. The pellet obtained from the 50 mL cultures was then resuspended in Tris Buffer Saline at an OD600 of 10. Once resuspended, the sample was cell lysed using sonication. Following sonication, the sample were span to separate the soluble and insoluble fragments form the whole cell lysate. 50 μL from each sample were obtained and stained with Laemmli reagent.
We proceeded on with purification of the soluble fragment using column chromatography containing Strep-Tactin resin. The process involved packing the column, equilibrating the resin and loading the soluble sample. Then a washing step was performed to remove any potential non bound nonspecific proteins. Then we eluted using competitive elution by loading BXT which competed with the mScarlet_DARPin929_StrepII for binding sites with the resin, thus detaching the protein of interest from the column. Finally, we recycled the column ready for future purifications. From each of the samples obtained during the procedure we obtained 50 μL to use for SDS PAGE and western blotting.
The mScarlet_DARPin929_StrepII-tag fusion protein has a total size of 45.1 kDa. As it can be observed from Figure 2 (a) and (b), BBa_K3111201 was highly expressed as it can particularly be observed from the thick bands at 45 kDa in lanes S, I and E2. We also observe an intense band at around 37 kDa, but still smaller than the one at 45 kDa, and smaller bands particularly at elution 2. We speculate that this is due to proteolytic cleavage of the fused mScarlet at specific sites generating smaller bands which are still linked to the DARPin929 and StrepII tag. This might interfere with accurate quantification of binding later on since some DARPins will not have a functional reported fused to them.
It is particularly interesting that in Figure 2 (b) the intensity of the band at 37 kDa is much higher compared to the image of the SDS PAGE. However, we concluded that the chemiluminescent reagent used for western blotting has higher binding affinities on smaller proteins fragments thus giving this false higher concentration of the smaller truncated protein.
Binding to HER2 receptor
The next step after protein purification, was to investigate the targeting peptides' binding effectiveness, which we are using on our drug delivery platform along with the T. maritima encapsulin. This involved culturing HER2 overexpressing SK-BR-3 breast adenocarcinoma cells, to which DARPin929 can bind to with nanomolar affinities, and observing binding and internalization using our fluorescent fusion protein mScarlet.
This involved culturing the cancer cells in T75 T-flasks until confluency was reached. After this, the cells were counted in order to passage them at the correct seeding density in the 6 wells plate where DARPin929 binding would be tested. The cells were left in the incubator at 37 °C and at 5% CO2 for 3 days in order to attach to the base of the flask and adapt. This allowed the appropriate morphology of the cells to be reached in order to optimise the final results of the experimentation.
Once the cells have attached, the DARPin929 hybrid protein was introduced to the media at concentration of 1,2 and 3 μM. These 3 different loading concentrations were tested to investigate the optimal concentration for improved binding and incorporation of the DARPin929, which would let us determine the possible required concentration of our novel drug delivery platform. To further incorporate the advice received from Dr. Yin Wu and Michael O’ Neil the cells were incubated at 37 °C for 1 hour to simulate the body temperature. For binding comparison, we also had a replicate of the plate at room temperature ca. 22 °C. Once incubation was completed, the medium was removed and cells washed with PBS to remove any unbound protein. 1 mL of DAPI, a nucleus-specific stain, was then added to help increase contrast and visualisation of the cells. Then plates were then visualised using EVOS inverted microscope.
Figure 3 and 4 indicate the images obtained in different wells post-incubation. These 2 experiments were done to optimise the condition of binding. While yet not conclusive, we could observe that fluorescence was observed both on the periphery and within the cells; sign that the DARPin gets internalised. DARPin_mScarlet, even at the lowest concentration showed better binding and uptake compared to mScarlet_DARPin. Nonetheless, while at lower intensity we could observe the same results using the required DARPin hybrid. Furthermore, 37 °C was more optimal for both constructs for binding and uptake and it would resemble the tumour microenvironment more closely . Finally, we concluded that 3 μM would be the best concentration for optimal binding.
In order to validate the binding specificity of BBa_K3111201, we conducted further control mammalian cell culture experiments by applying the optimal concentration of 3 μM of the fusion protein found throughout our optimisation experiments. These experiments were done to firstly make sure that proteins without fused DARPins would not bind or get uptaken by the cells. This was done by applying rTurboGFP at the same concentration. Secondly to observe the specificity of DARPin and subsequently our drug delivery platform we then applied the DARPin_mScarlet fusions, on Mesenchymal stem cells (MSCs) which do not express HER2 receptors. Additionally, we wanted to observe the fate of encapsulins once applied on cells. Thus we manufactured encapsulins without DARPins (BBa_K3111104) but surface displayed iLOV, a cyan fluorescent protein and applied it at the same concentration as the previous constructs on SK-BR-3 cells. Moreover to better replicate the tumour microenvironment we incubated mScarlet_DARPin929 with SK-BR-3 cells at hypoxic environment of 2% oxygen to observe whether binding was affected.
From Figure 5, we observed no fluorescence thus no binding or uptake of both rTurboGFP and Thermotoga maritima encapsulin fused with iLOV. Thus, we confirm that in the absence of DARPins fusion proteins cannot bind or get internalised. Moreover, we observe no red fluorescence on the MSCs indicating the specificity of DARPin only for HER2 receptors, thus confirming the potential for targeted delivery platforms by using DARPin. From Figure 6 we could observe reduced binding at hypoxic conditions compared to normoxic condition as observed in Figure 5.
To obtain greater accuracy of binding percentage we proceeded with flow cytometry. Thus, we dislodged the cells from their growth wells using EDTA, while not affecting the binding of the DARPin and the HER2 receptor, and then used the sample for flow cytometry. We used the flow cytometer (BD Accuri C6 Plus) configured to observe the fluorescence within the mScarlet and CFP regions.
The flow cytometry data allowed precise quantification of the population of cells that has the DARPin929 bound on the surface. In the plots of Figure 7, whatever is on the right-hand side of the red line shows binding and correspondingly whatever is on the left, no binding. Based on Figure 7 (a) & (d) we observe that DARPin929_mScarlet has better binding efficiency with 29.8% compared to mScarlet_DARPin929 with only 19.6%. Clearly it can be noticed, that there is no unspecific binding when no DARPin is present as in the cases of rTurboGFP and T. maritima encapsulin fused with iLOV as well as when the DARPin fusion proteins have no receptor to bind to as in the case of Figure 7 (b) and (e). It was interesting to notice that binding percentage of mScarlet_DARPin929 at hypoxic condition observed in Figure 8 does not vary significantly to the one obtained in normoxic condition as observed in Figure 7 (a). This allowed us to hypothesize that possibly the tumour hypoxic microenvironment will not affect the potency of our drug delivery platform.
Furthermore, we wanted to investigate the rate of internalisation by observing the change in binding percentage at different incubation times. Therefore, we proceeded with flow cytometry of the SK-BR-3 samples obtained at 15 and 30 min incubation.
While we would expect binding to reduce over the span of the different incubation times, this was not observed from the flow cytometric analysis in figure 9. We could observe that over time the binding of BBa_K3111201 is increasing. We speculate that internalisation happens simultaneously as it can be observed in the images of Figure 9 where the fluorescence is observed across the cytoplasm adjacent to the DAPI-stained nucleus. However, to obtain more accurate data regarding this, future experiments would involve lysing the cells to release the fluorescent hybrid DARPin929 protein.
Conclusion
Therefore, this data allowed us to conclude that we are able to fuse DARPin929 on the C-terminus of another protein while retaining its' functionality. This revelation allowed us to design the most important parts of our drug delivery system - BioBricks BBa_K3111501 and BBa_K3111502.
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