# Designing a Cleavable Cell Surface Protein for Cytotherapy and Drug Delivery Applications
## 4 Discussion  
De-novo design of proteins with novel functions is an extremely complex computational technique. A potentially simpler method is fusion protein design which uses naturally occurring protein domains in novel combinations to design new functions [26]. This method has been used to design a wide array of synthetic proteins with novel combinations of functions [24; 26; 27]. Here, this method was used to combine the substrate binding function of a novel monostreptavidin protein with the ability of uPAR to be cleaved by uPA in order to design a system that could release a payload in response to uPA activity. The fusion protein method allowed the design of a protein that could: (1) bind biotinylated cargo; (2) be expressed on the cell surface; and (3) release the cargo in response to uPA activity by using previously designed or naturally occurring domains from other proteins. This facilitated the design of a novel protein with novel activity with significantly less input than de-novo methods.  
One challenge in fusion protein design is designing peptide linkers between the domains that allow each domain to fold correctly and interact as expected [24]. Placing protein domains next to each other in a fusion protein runs the risk of having the domains interact in unexpected ways leading to a misfolded and/or inactive protein. To reduce the chance of this, in the protein designed here, relatively rigid \(\alpha\)-helix spacers were used to insulate the domains from each other and to allow uPA access to the cleavage sequence.  
In the expressed mSA protein, each domain functioned as expected. The PLAP sequence caused the mSA protein to be expressed on the cell surface (Figure 6). The modified mono-streptavidin domain was able to efficiently bind biotinylated moieties (Figure 4). The uPA consensus sequence and uPAR domain was able to be cleaved by uPA (Figure 5) allowing release of the payload.  
The purpose in designing this protein was to allow a payload to be loaded onto the surface of tumor-homing cells and then be released in the elevated uPA activity often found  
Figure 6: Confocal images of labeled cmSA-C17.2. (**A**): cmSA-C17.2 labeled with biotin (5-fluorescein). (**B**): cmSA-C17.2 labeled with PLGA-rhodamine-biotin nanoparticles.  
in tumors. The biotinylated fluorescein was an example of a small-molecule drug-like molecule. This system could be used to target small-molecule chemotherapies (e.g., doxorubicin, cisplatin, vincristine, etc.,) to uPA producing tumors using modified tumor-tropic delivery cells. This system could allow for a simple method for loading delivery cells with a therapeutic and then releasing that therapeutic at the target location.  
One unexpected result in the system was the internalization of nanoparticles bound to the cmSA protein. C17.2 cells are not known to be significantly phagocytic, nor, as neural progenitor cells, would they be expected to. WT C17.2 behaved as expected and did not demonstrate any significant interaction with the PLGA nanoparticles during incubation. cmSA-C17.2 showed evidence of binding to the PLGA nanoparticles as expected, but then, unexpectedly, appeared to internalize the nanoparticles. This may indicate that stably binding a nanoparticle in close proximity to the cell membrane can cause internalization in otherwise hard to transfect cells. This may suggest alternative uses for mSA genes in internalizing cargos such as DNA, nanoparticles, etc., in cells that would not normally internalize them.