Team:UCL/Validate
Validate: M.xanthus Encapsulin and DARPins
Introduction
We first wanted to explore and validate the modularity of our drug delivery platform, and investigated the feasibility of using different types of encapsulins, including ones that have not been worked with before within iGEM. We expressed, purified, and studied the Myxococcus xanthus encapsulin, which is slightly larger than that of T. maritima. Moreover, we studied the encapsulin’s structural and physiological properties to obtain insight on the stability of the encapsulins and its feasibility as a potential drug delivery vehicle.
The second part of our validation involved testing our chosen binding peptides, DARPins. It was crucial as it dictated the feasibility of our targeting mechanism. We studied the expression of DARPin929, which also had not been previously characterised as part of iGEM. DARPins are genetically engineered antibody mimetic proteins that exhibit highly specific and high-affinity binding to their target protein. DARPin929 has previously been shown to bind very stringently to HER2 receptors (down to picomolar affinities)(1,2) and DARPin drug canditates are currently on different clinical trial phases (3).We wanted to investigate two major elements: 1) Whether C terminal fusion of DARPin would affect binding to HER2 receptors and 2)study the possibility of targeted binding.
Following expression of DARPin929 in E.coli, we proceeded with mammalian cell experiments to investigate these elements. Using flow cytometry and fluorescence microscopy, we measured binding of DARPin929 on HER2 expressing SK-BR-3 breast adenocarcinoma cells. To study how the tumour microenvironment could affect the affinity of binding we also included tests on binding at tumour physiologically relevant oxygen levels.
Experimental Approach and Results
M.xanthus Encapsulin
We successfully expressed and purified the Myxococcus xanthus encapsulin monomer. We performed an SDS page on FLAG purified protein, and could observe a band just above 35 kDa in the un-purified cell lysate (lane L) and purified elution lanes (E2-E4) as shown in Figure 1. This band size does vary slightly to the expected 33.05 kDa size calculated from the protein sequence, however we think this may be due to common variability in motility of proteins of the same size (4).
In order to confirm the encapsulin monomers were properly assembling into shells, we carried out transmission electron microscopy (TEM) on the purified sample with the help of Mark Turmaine from the division of biosciences EM core facility at UCL. We could observe a concentrated sample of round-shaped correctly assembled encapsulins as shown in Figure 2. While the majority of the assembled encapsulins were within the size range of 30 nm, we could also observe smaller variants (picture within the red circle). This is consistent with the literature (5).
DARPins
We also were able to successfully clone, express and purify both constructs BBa_K3111201 (mScarlet_DARPin929) and BBa_K3111202 (DARPin929_mScarlet). We wanted to test both configurations of DARPin (with a fluorescent protein bound to the N terminal and C terminal). This is because the DARPin-mScarlet construct has been tested before in literature, but the mScarlet-DARPin construct closer resembles our future encapsulin-DARPin final construct. Therefore we wanted to make sure the terminal to which the DARPin is bound would not hinder Her2 binding ability.
From an SDS PAGE of both constructs shown in Figure 3, we were able to observe that they were the expected size of 45.1 kDa. Also visible from the figure both parts were highly expressed as can be observed fomr the thick bands at 45kDa in the solluble (S), insolluble (I) and purified (E1, E2, and E3) lanes.
Following successful expression and purification of our DARPins we tested their binding ability to our Her2 expressing breast cancer cell line, SK-BR-3. We first imaged the fluorescently tagged DARPins binding to SK-BR-3 cells and our non-Her2 expressing control mesenchymal stem cell (MSC) line using confocal microscopy as shown in Figure 4. We observed binding of both DARPin constructs specifically to our breast cancer cell line. We also tested their binding under hypoxic conditions similar to those found within tumours, and found the DARPin constructs were able to bind to our breast cancer cells as shown in Figure 5.
In order to quantitatively characterise binding, we ran our cells incubated with our fluorescently tagged DARPins in a flow cytometer. Figure 6 shows flow cytometry results recorded after incubating our breast cancer cell line or the MSC control with 3uM of the DARPin constructs for 1 hour.
We can observe that while a 10% higher binding efficiency was detected with the previously documented DARPin-mScarlet construct, we still observe considerable binding with the mScarlet-DARPin construct. Thus we will be still able to direct our drug delivery platform to HER2 overexpressing cells. It was interesting to notice that the binding percentage of mScarlet-DARPin in hypoxic condition observed in Figure 7 does not vary significantly to the one obtained in normoxic condition as observed in Figure 6. This allows us to hypothesize that the hypoxic tumour microenvironment may not affect the binding ability of our drug delivery platform.
We also validated the binding specificity of DARPin929 using untargeted fluorescent markers as control. We observed no fluorescence, and no unspecific binding or uptake, of both rTurboGFP and Thermotoga maritima encapsulin fused with iLOV, a cyan fluorescent protein as shown in the right hand column of figure 5. Thus, we confirm that in the absence of DARPins, the fluorescent proteins will not bind or get internalised. Moreover, no binding was detected on the non-Her2 expressing MSCs indicating the specificity of the DARPins for HER2 receptors and confirming the potential for the targeted aspect of our delivery platform.