Computational Modeling

  Prior to designing our fusion proteins, we investigated how FGF-2 and NT-3 would change structurally after they formed a fusion protein with HIRMAb. We used PyMol as a molecular visualization system and used Ramachandran Plot to calculate structural deviation in wild-type FGF-2/NT-3 and HIRMAb-FGF2/HIRMAb-NT3.

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  In the case of FGF-2 N-terminal residues (Methionine-alanine-alanine) binds with the HIRMAb (Trojan Horse) due to which structural changes occur in protein. The structure changes its symmetry which also changes its functional ability. For example, the wild-type FGF-2 structure contains beta sheets with a small part of α-helix at the N-terminal and C-terminal. Whereas in the case of HIRMAb-FGF2 tertiary structure of FGF2 changes its symmetry as well the number of βsheets was reduced, and the α-helix unit was observable. More loop region is also present which changes the surface orientation of the heparin-binding region, therefore, by joining the HIRMAb sequence with FGF-2 symmetry of the structure changes as well as its function.

  In the case of the HIRMAb-NT3 (Trojan horse) structure, beta sheets and alpha helix structures were present. However, in wild type, NT-3 structure only beta sheets were shown that means by adding HIRMAb sequence with NT-3 structural changes occur in protein. Although α-helix were unstable due to fewer hydrogen bond interactions. Mostly α-helix were involved in water-based interaction therefore, parallel beta sheets are more stable than antiparallel beta sheets. Thus, the wild-type NT-3 structure contains only antiparallel beta sheets which means it is not a much more stable structure having 2.60 Å resolution. In another case, HIRMAb-NT3 structure contains alpha helix and beta sheets which means by adding the HIRMAb sequence with NT-3 sequence its folding capacity increases and now the territory structure contains both secondary structures. Therefore, structural change in protein folding will change its function as well. Although, experimental validation including simulations was required to confirm the stability of α-helix.

Constructing Plasmids

  After cloning our plasmid constructs for our Trojan horses and some of their cell receptors, we used restriction enzymes to cut our plasmids, and ran the results on a gel. The restriction enzymes BamH1 and EcoR1 were used to cut the receptor plasmids, while the enzymes Notl and Xhol were used to cut the Trojan horse plasmids.

  We modeled our expected results on Benchling. Both our model and gel had similar results.

Transwell Assay

  HCMEC/D3 cells were plated on transwell inserts and cultured until full confluency. LiCl was added to the culture media to stimulate the formation of tight junctions in order to model the Blood-Brain Barrier. Different HIRMAb proteins were added to the transwells to see if they would be transported by the HCMEC/D3 cells to the other side of the Transwell, as this would happen in vivo from the blood stream into the brain. In order to detect if the Trojan horses were transported through the Transwell, we ran a Western Blot to detect them on the "brain" side, or the lower comaprtment of the Transwell. We saw no bands from our Western Blot, which early on, seemed to indicate that our Trojan horse was added to our transwell insert's upper compartment but was unable to cross the Blood-Brain Barrier, a theory we disputed later.

Interaction Assay

  CHO-K1 cells were transfected with the plasmids expressing our engineered Trojan horses (HIRMAb-HC, HIRMAb, HIRMAb-FGF2, HIRMAb-NT3) and receptors (HIR-eGFP, FGFR2-eGFP, NTRK3-eGFP). The media collected from these cells 24 hours after transfection contains the Trojan horses proteins and, after concentration using centrifugal filters, were used to test the ability of the Trojan horses to bind to their respective receptors.

  12 wells seeded with CHO-K1 flasks were transfected with our 3 receptor plasmids (4 each). These cells will express the receptors on their surface and will be used to see whether the Trojan horses bind to their respective receptor.

  We added the media containing the concentrated Trojan horse proteins to the cells expressing the different receptors. Cells and any molecular interaction were fixed with paraformaldehyde. To determine that our Trojan horse binded to its receptor, we added a secondary anti-human IgG antibody attached to a red dye. The receptors are fused to eGFP and cells expressing the receptors are therefore easily identified due to their green fluorescence under a fluorescent microscope. Any interacting between the Trojan horse and its receptors will show as co-localization of red fluorescence on top of cells also positive for the green fluorescence.

Co-transfection of HIR-eGFP with HIRMAb under green light.

Co-transfection of FGFR2-eGFP with HIRMAb-FGF2 under green light.

Co-transfection of NTRK3-eGFP with HIRMAb-NT3 under green light.

  The transfections for our receptors were consistent with our expectations for the experiment. The eGFP for HIR, FGFR2, and NTRK3 exhibited bright green luminescence in their wells.

Co-transfection of HIR-eGFP with HIRMAb under red light.

Co-transfection of FGFR2-eGFP with HIRMAb-FGF2 under red light.

Co-transfection of NTRK3-eGFP with HIRMAb-NT3 under red light.

  We expected the transfections with our Trojan horses to co-localize with the receptor transfections and produce a green-red illuminescence in some of our wells. However, none of our wells displayed a green and red double signal. We ran the experiment again with a higher concentration of our secondary antibody from 1:1000 to 1:200, but still received the same results. At this point, we considered the possibility that our Trojan horses might not have ever been there in the first place, that our CHO-K1 cells were unable to produce our fusion proteins and secrete them into media. Thus, we ran a Western Blot on our Trojan horses. We found that there were no bands produced, similar to the Western Blot from our transwell assay. This suggested that our CHO-K1 cells had difficulties expressing our fusion protein after transfection with our recombinant plasmids. While all of our plasmids were confirmed with restriction digests, none of them were sequenced. Thus, it is possible that our plasmids acquired a mutation early on, like one that introduced a stop codon into the protein's reading frame.

  In conclusion, our team was able to use computational modeling to initially identify the changes that would occur in NT-3's and FGF-2's protein structure if they were fused to HIRMAb. Our results showed that NT-3 would be structurally more stable as part of the fusion protein than its wild type version, while FGF-2 would be less stable than its wild type version. We engineered our Trojan horse and receptor plasmids using PCR and Gibson cloning, and ran our final plasmids on gels with restriction enzymes to validate we were working with the right ones. After running a transwell assay, we found from our Western Blot that our proteins were not in the lower compartment. The results for the interaction assay, while unexpected, do not provide conclusive evidence that HIRMAb-FGF'2 or HIRMAb-NT3's components can bind or can not bind to their respective cell receptors. From the results of the Western Blot, we found that our CHO-K1 cells were likely unable to produce our Trojan horse proteins, which explained why our final assays both failed.