Team:NYMU-Taipei/Design

Design

Overview

Our design idea is to capture and denature the SARS-CoV-2 using our customized protein. We chose the receptor-binding domain (RBD) of human angiotensin-converting enzyme 2 (hACE2) as the capturer of SARS-CoV-2. The spike protein on the surface of the virus could bind with hACE2 (RBD) and enter host cells via this mechanism. Moreover, we attached a protease, which is aminopeptidase P(pepP), to the bottom of the hACE2 (RBD) to denature the spike proteins captured. By doing so, we could not only create an effective protective surface against SARS-CoV-2 but also deactivate the virus we captured simultaneously. The following paragraph will get into more detail about the procedures of our experiment.

Gel that contains Cu2+ ions is created and applied on a surface

Capturing

It has been proven that the receptor-binding domain of the spike protein of SARS-CoV-2 can bind to the cell receptor ACE2 when infecting human beings (see figure below). By utilizing this fact, we designed for a part of our construct to be a synthetic ACE2 receptor binding domain to capture the virus.

Attach the Ace2 RBD construct to our gel
SARS-CoV-2 Spike protein binds to gel

Detection

We designed a tiny box which can detect if there is SARS-CoV-2 in the user’s breath. Inside the box, there is a piece of gel and ACE2. His tag is added to hACE2 construct so that ACE2 can chelate with Co2+ on our gel, and thus can be presented on the surface of our gel. To achieve detection, we attached a red fluorescent protein(RFP) to our designed ACE2 construct since RFP is visible to the naked eye. This design would show if one’s breath contains SARS-CoV-2 visibly. How do we use this product? Firstly, we would let the user blow in the box through a hose on it. After blowing in our device, the gel with hACE2 would capture spike protein as mentioned above, if any. Next, we add ACE2 with RFP to the box, and then gently shake the box for about three minutes in order to let all the spike proteins on the virus bind tightly to the ACE2 attached with red fluorescent protein. Afterwards, we use wash buffer (PBST) to wash off ACE2 without spike binding. Lastly, only ACE2 with spike would be left on the gel, and RFP would show if one is infected with SARS-CoV-2 and attain detection. More details on the design of the box can be found in the Application page on our wiki.

Captured Spike protein is covered by our hACE2-RFP construct
hACE2-RFP construct attached to Spike protein emits light for detection
Unattached hACE2-RFP construct washed away by buffer
SARS-CoV-2 is captured and detected

Inactivating

We want to inactivate SARS-CoV-2 by preventing the virus from entering our cells. To achieve this goal, we came up with an idea of using a protease to cut the spike protein of SARS-CoV-2. Past research has indicated that Xaa-Pro aminopeptidase, also known as pepP, could be the appropriate protease to inactivate spike protein, and checking through protein interaction modeling, our final choice was to add pepP to our ACE2 construct. Ideally, the ACE2 part of our construct would capture the virus and the pepP part would cut spike protein into many fragments. This would damage spike protein, thus efficiently making the virus unable to infect humans since SARS-CoV-2 relies on spike protein to enter host cells. Eventually, we applied our design to face masks so that we can be free from worries about contacting virus when touching face masks. Further details of application on face masks will be shown in Application page of our wiki.

Spike protein comes into contact
Spike protein binds with Ace2 RBD and our aminopeptidase takes effect
Spike protein is cleaved by our aminopeptidase
Spike protein successfully inactivated
Xaa-Pro aminopeptidase (pepP)

The gene of Xaa-Pro aminopeptidase belongs to a network of genes which facilitate stress-induced mutagenesis (SIM) in E. coli K12. The length of Xaa-Pro aminopeptidase is 1326bp / 441aa. The active site residues, His243 and His361 are required for catalysis; His350 and Arg404 are involved in the proline substrate specificity; His350 and Arg404 are involved in the proline substrate specificity; Tyr387 and Arg404 are important for substrate hydrolysis. For more 3D structure details, please visit RCSB.

References
  1. Lan, J., Ge, J., Yu, J. et al. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature 581, 215–220 (2020).
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  5. Graham SC, Lilley PE, Lee M, Schaeffer PM, Kralicek AV, Dixon NE, Guss JM (2006). “Kinetic and crystallographic analysis of mutant Escherichia coli aminopeptidase P: insights into substrate recognition and the mechanism of catalysis.” Biochemistry 45(3);964-75.
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  7. Jao SC, Huang LF, Hwang SM, Li WS (2006). “Tyrosine 387 and arginine 404 are critical in the hydrolytic mechanism of Escherichia coli aminopeptidase P.” Biochemistry 45(6);1547-53.
  8. Kuhn ML, Zemaitaitis B, Hu LI, Sahu A, Sorensen D, Minasov G, Lima BP, Scholle M, Mrksich M, Anderson WF, Gibson BW, Schilling B, Wolfe AJ (2014). “Structural, kinetic and proteomic characterization of acetyl phosphate-dependent bacterial protein acetylation.” PLoS One 9(4);e94816.
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  11. Yoshimoto T, Murayama N, Honda T, Tone H, Tsuru D (1988). “Cloning and expression of aminopeptidase P gene from Escherichia coli HB101 and characterization of expressed enzyme.” J Biochem (Tokyo) 104(1);93-7.
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  13. Yoshimoto T, Orawski AT, Simmons WH (1994). “Substrate specificity of aminopeptidase P from Escherichia coli: comparison with membrane-bound forms from rat and bovine lung.” Arch Biochem Biophys 311(1);28-34.
  14. A T Orawski, J P Susz, W H Simmons (1987). “Aminopeptidase P from bovine lung: solubilization, properties, and potential role in bradykinin degradation.” Mol Cell Biochem. Jun;75(2):123-32.

A Project conducted by the 2020 NYMU iGem Team
nymuigem2020@gmail.com
@wetlabtenpeaple
NYMU iGEM Team @igemnymutaipei
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