Difference between revisions of "Part:BBa K3111011"

Revision as of 10:58, 11 October 2019

DARPin929

This part encodes DARPin929 – a binding protein specific to HER2 receptor, that is similar to an antibody in terms of efficiency and specificity. However, due to its stability and ability to be expressed in bacteria, this DARPin can be used in a wider variety of applications (1). The part has had its start and stop codons removed for flexible fusion to N or C terminus.

Sequence and Features

Usage and Biology

Natural Ankyrin Repeat Proteins are versatile binding molecules which can bind to specific targets and subsequently trigger various molecular mechanisms (e.g. enzyme inhibition or protein anchoring) (2,3). Aiming to manipulate the immunologic potential of said repeat proteins for therapeutic applications, in 2003, Plückthun et al. genetically engineered designed DARPins (4). DARPins are small (14-21 kDa) single-domain binding molecules derived from natural ankyrin repeats (5). As natural repeat proteins, DARPins comprised a several structural motifs which stack to form the repeat protein domain (4,6). The assembly of highly expressed, stable and soluble DARPins happened by production of DARPin libraries using ribosome, phage display etc. indicated that these binding molecules had the potential to circumvent the limitations of monoclonal antibodies and empower new therapeutic approaches (4,6).

DARPin929 has been one of the most frequently used to evaluate probes for molecular imaging. It particularly targets human epidermal growth factor 2 (HER2) whose overexpression is associated with breast cancer and gastroesophageal cancer. Has been shown to bind down to nanomolar affinities to its target (7,8). DARPins have been shown to be very stable and show less tendencies for aggregation, unlike single chain variable fragments since they do not possess cysteines.

Functionalisation

BBa_K3111011 has been used along with BBa_K3111021 for initial investigation of binding to SK-BR-3 HER2+ breast adenocarcinoma cells. Moreover, it was fused with BBa_K3111003 and co-expressed with BBa_K3111032 to create the drug delivery vehicle containing cytotoxic cargo (BBa_K3111502).

Structural modelling of DARPin929 fusion to encapsulins

We suspected that fusing DARPin929 to the outer surfaces of T. maritima and M. xanthus encapsulins may result in steric clashes between encapsulin-DARPin dimers, thus impeding or preventing the assembly of the full encapsulin capsules. In order to assess the feasibility of fusing DARPin929 to either encapsulin, we created protein models using PyMol (GUI and manual edits), Discovery Studio (structure quality), GROMACS (energy minimisation of created dimers) and atomium (rebuilding the full encapsulin capsules).

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As a result we conclude that fusing DARPin929 to the T. maritima encapsulin is more readily feasible. We demonstrate this fusion in BBa_K3111501.

Experimental Results

Experiments showing the properties of BBa_K3111011 were carried out using composite parts BBa_K3111201, BBa_K3111202, BBa_K3111501, BBa_K3111502 and BBa_K3111503.

Expression and Purification

Fusion to BBa_K3111003

Confirmation of assembly

Using composite part BBa_K3111501 we investigated the ability of DARPin929 to be fused onto T. maritima encapsulin without hindering its assembly. We first demonstrated that this fusion protein would express and purify without cleavage by running it on an SDS gel (Figure 1). From this we could observe that the encapsulin became strongly insoluble after the addition of DARPin929, however, a small fraction of it could still be purified, as seen in lane 2 and 3 on the gel (~51 kDa). We did not observe any smaller bands (besides host cell protein at ~15 kDa), indicating that the DARPin was not cleaved off.

Figure 1: SDS PAGE of T.maritima encapsulin monomer_DARPin929_StrepII; S: Cell lysate Soluble Fragment, I: Insoluble cell lysate fragment, L: Load, W: Wash, 1-3: Elution 1-3

To observe assembly under non reducing environment, we concentrated the purified sample from elution 2 and used it for Transmission Electron Microscopy imaging and non-reducing PAGE gel.

Figure 2: a) Transmission Electron Microscopy of assembled T.maritima encapsulin_DARPin929_StrepII; Scalebar: 50 nm, b) Native PAGE gel of assembled T.maritima encapsulin_DARPin929_StrepII.

Figure 2 (a) shows the TEM image obtained. We could clearly observe assembled encapsulins thus concluding that although expression of the protein was mostly insoluble, the soluble fragment contained fully assembled encapsulins. The native PAGE gel observed in Figure 2 (b) let us confirm that the DARPin did not get cleaved-off during assembly, as we observed a significant increase in band size from the TmEnc lane to the TmEnc_DARPin929.

After these observations we decided that assembly was not very critically affected and moved to cargo loading in part BBa_K3111502.

Impact of DARPin fusion on cargo loading

Furthermore, we investigated the impact of fusing DARPin929 on cargo loading into the encapsulin by evaluating two different DARPin display strategies. First, BBa_K3111502 showed the loading of mini singlet oxyged generator (miniSOG) into an encapsulin which has a DARPin on every monomer, whereas, BBa_K3111503 had a mixture of encapsulin monomer with and without a DARPin.

Figure 3: Quantification of T. maritima encapsulin loading. Error bars show 95% confidence interval.

Figure 3 shows the cargo loading capacity obtained from producing the multicomponent drug delivery platform using the aforementioned cloning strategies. Loading capacity for BBa_K3111502 was estimated to be 13.61+-0.36 molecules of miniSOG. In contrast, BBa_K3111503 was shown to be loading 8.19+-0.21 miniSOG molecules per encapsulin. We hypothesise that this difference is caused by the surface display of DARPins slowing down the assembly of encapsulins and giving the targeting peptide present on miniSOG more time to form hydrophobic interactions to the inner shell of encapsulin monomers. Analysis on the methodology used to calculate the number of loaded cargo proteins can be found on BBa_K3111402 registry page.

Evaluation of binding

Performance in encapsulin-based drug delivery system