Part:BBa_K3237013
Insulin Receptor Protein under Constitutive Expression
The insulin receptor protein is a regulator of glucose transport in and out of cells by its interaction with the presence of insulin protein[1]. This construct was designed with a N-terminal His tag for affinity purification and was placed under constitutive expression as seen with part BBa_K3237013. This part was created in order to perform binding assays and to confirm proper processing/activity of insulin constructs made by the 2019 University of Lethbridge iGEM team. The part was also optimized for expression in E.coli to obtain the quickest yields as a opposed to microalgae expression which our project proposes for insulin production.
Protein Purification
Figure 1: 16.5% Tricine gel of the Nickel affinity chromatography of the receptor protein run on a gravity column. The protein is shown at the 10kDa mark. However, the insulin is dimerizing and tetramerizing causing the larger bands on the gel.
Figure 2: A 16.5% tricine gel of Size Exclusion Chromatography of receptor protein. The last elution has thicker band at 25kDa which we suggested that is our insulin protein.
Figure 3: A size exclusion chromatogram of the receptor protein. Peaks 4 and 5 contain the target protein.
MicroScale Thermophoresis
with the successful purification of the insulin receptor we ran a MicroScale Thermophoresis (MST) assay to see if our insulins were able to bind. Unfortunately we were only able to test the SCI57 construct (BBa_K3237018).
Figure 1: A time scale graph showing the MicroScale Thermophoresis assay indicating protein-protein interactions between the mRFP1-SCI57 protein and the insulin receptor. The blue data indicates fluorescence reads with just the mRFP1-SCI57 construct at 500nM concentration. The green data represents the samples containing the mRFP1-SCI57 and insulin receptor at 500nM concentration and ~17,000nM receptor respectively. The increase in fluorescence reads with the mRFP1-SCi57 + insulin receptor protein indicates a binding event between the two proteins.
NYC Empire State 2022 - Human Insulin Receptor and its Interaction with Human Insulin Receptor Monooclonal Antibody
The NYC Empire State team designed a biological Trojan horse that consisted of two proteins. The first was Human Insulin Receptor Monoclonal Antibody (HIRMAb), which can interact with the HIR (Human Insulin Receptor) on endothelial cells making up the Blood-Brain Barrier in a process known as receptor-mediated transcytosis. This process moves HIRMAb, and anything fused to it, across the Blood-Brain Barrier and into the brain. The second is a "therapeutic protein," either FGF-2 (Fibroblast Growth Factor 2) or NT-3 (Neurotrophin-3). Once these proteins enter the brain, FGF-2 binds to FGFR2 (Fibroblast Growth Factor Receptor 2), while NT-3 binds to NTRK3 (Neurotrophic Receptor Tyrosine Kinase 3).The NYC Empire State team ordered the sequence for the sequence for HIR attached to eGFP from Twist Bioscience. We cloned the plasmid through PCR and Gibson cloning, and ran the result on a gel with restriction enzymes. Our gel's results conformed with our expectations from modeling the gel on Benchling.
Figure 1: Results of running HIR-eGFP on a gel with restriction enzymes compared to expected results on Benchling
After these results, we transfected our plasmids into CHO-K1 cells using jetOPTIMUS. To test if our two fusion proteins, HIRMAb-FGF2 and HIRMAb-NT3, as well as HIRMAb and HIRMAb's heavy chain could cross the Blood-Brain Barrier, we designed a Transwell assay. We set up 5 conditions (untreated, HIRMAb-HC, HIRMAb, HIRMAb-FGF2, HIRMAb-NT3) with 2 samples each for a total of 10 transwells. Our team cultured a layer of confluent HCMEC D3 cells on a transwell insert. These cells model the human Blood-Brain Barrier. To more closely model the tight junctions of the actual Blood-Brain Barrier, we treated these cells with lithium chloride. After adding our different proteins into the upper compartments of our transwells, we waited for time intervals of 30, 60, and 120 minutes to collect media from the lower compartment. We ran a Western Blot on the results. However, our Western Blot did not have any bands on it. At first glance, it would seem that our Trojan horses were not able to cross the Blood-Brain Barrier. But after running the same Western Blot on the original media from our CHO-K1 cells, we saw the same results. This might indicate that our original CHO-K1 cells were not able to successfully produce and secrete our Trojan horse into the media.
Figure 2: Model of transwell assay
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |