Part:BBa_K3111104
T. maritima encapsulin (6-His) fused to iLOV with a StrepII-tag
This BioBrick was used to initially explore the possibility of expression and assembly of an encapsulin with a protein on its surface. Therefore, iLOV, a constitutively fluorescent cyan fluorescent protein was cloned downstream of the encapsulin monomer coding sequence, to allow its surface display. This was followed by expression studies and protein analysis. Additionally, it has been used as a control sample to study the fate of encapsulins without HER2 targeting peptide DARPin929 (BBa_K3111011). For this it was incubated with SK-BR-3 breast adenocarcinoma HER2+ positive cells. This showed that encapsulins are not uptaken by SK-BR-3 in the absence of surface displayed binding peptides, as fluorescence was not detected in the cell culture post-incubation.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 171
Illegal BglII site found at 586
Illegal BamHI site found at 950 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 520
Illegal SapI.rc site found at 551
Composite Part Design
iLOV, a constitutively fluorescent cyan fluorescent protein was cloned downstream of the encapsulin monomer coding sequence (BBa_K3111003), to allow its surface display. A StrepII-tag was added at the C-terminus of iLOV protein to allow for column purification. While the original purpose of this construct was to test fusion of protein to he C-terminus of T. maritima encapsulin, the fluorescent properties allowed us to use the sample to observe the fate of the encapsulin molecules without DARPin929 targeting peptide once incubated with SK-BR-3 cells using confocal microscopy.
Experimental Results
DNA Analysis and Cloning
We started by cloning BBa_K3111104 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 2613 bp and 956 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_K3111104 sequence coding for T. maritima encapsulin (6-His) fused to iLOV with a StrepII-tag 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_K3111104 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_K3111104. 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 BBa_K3111104 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 TmEncH_iLOV 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.
The TmEncH_iLOV fusion protein has a total size of around 46 kDa. In Figure 2 we can see this band in both the soluble and insoluble fragments. The intensity of the band however is thicker in the insoluble fragment thus the fusion protein is majorly insoluble. However, this band was also observed in both elution 1 and 2 (surrounded by orange box) thus indicating that the fusion protein from the soluble fragment was purified. However, we noticed that elution 1 was contaminated from the wash, thus for the rest of the experiments we decided to use samples from elution 2.
Further spectrophotometric analysis indicated fluorescent emission from the sample thus we concluded that the T. maritima encapsulins with surface displayed iLOV successfully expressed.
Studies of encapsulin uptake by SK-BR-3 cells
As previously mentioned, to observe the fate of encapsulins without conjugated DARPin929, we used a sample from elution 2 observed in Figure 2 and carried out mammalian cell culture studies using HER2 overexpression SK-BR-3 cells. This was done as a control along with incubation of the cells with BBa_K3111021, mScarlet_DARPin929, used to study binding efficiency and specificity of ours drug delivery platform targeting peptide, namely DARPin929.
This involved culturing cells SK-BR-3 cells at a density of 100,000 cells/cm2 and applying the purified protein at a concentration of 3 μM. After one-hour incubation with cells at 37 °C, 20% O2, 5% CO2 the cells were washed with PBS to remove any potential unbound substances in the growth medium, stained with DAPI, a nucleus stain, and the cells imaged under the EVOS FL microscope.
Figure 3 shows the images obtained after confocal microscopy. mScarlet is red fluorescent, while iLOV green. It could be observed that with mScarlet_DARPin929 the SK-BR-3 cells were red on the circumference but not only. Regions surrounding the blue-stained nucleus were also red. These two results indicated that the fusion protein mScarlet_DARPin929 was both binding and internalised by the cells. In comparison, when the cells were incubated with T. maritima encapsulin with surface displayed iLOV, no green fluorescence was observed. This let us conclude that in the absence of DARPin929 fusion proteins encapsulins cannot bind or be internalised on the HER2 overexpressing cells and that the use of DARPin929 on our platform will allow specifically targeted drug delivery.
To obtain greater accuracy results, we dislodged the cells from their growth wells using EDTA and used the samples for flow cytometry for detection of bound fluorescence. The results can be observed in Figure 4. As observed in the sample with the encapsulin there is essentially nothing bound compared with the mScarlet_DARPin929 were at least 20% fusion proteins are bound. Unfortunately internalisation could not be detected because that would require cell lysis experiments. These results reconfirmed that there is no binding in the absence of DARPin929, thus showing the specificity and targeting of DARPin929 on HER2 receptors. Using this data we will move onto cytotoxicity assays with our multicomponent DARPin-encapsulin based drug delivery platform.
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