Team:NYMU-Taipei/Engineering

Engineering

The very start goal of our project is to eliminate the suspending COVID-19 virus particles in the air. Through synthetic biology, we could achieve specific capture and inactivation. This could be helpful in highly dangerous or intricate working place, such as the custom or IC chip factory, since the air flow needs extra protection.

Phase One Design (March to July)

Through researching previous experiments, we found out that huamn receptor hACE2. This could be used to capture the flowting virus in the air to fulfill our need. Next, in order to inactivate the virus, we cameup with an idea to malfunction the virus with a protease. According to documentation, COVID-19 virus enter human cell through the binding between spike protein and human receptor hACE2. After that, a human membrane protein called the TMPRSS2 will cut spike protein on S1/S2 domain, enable the virus to enter human cell through membrane funsion.

Thus, our phase one design is to construct a fusion protein, which is consist of hACE2, a linker and a selected protease. The protease is choosing according to peptidase database “MEROPS”. We wish to find something only reacts with spike protein rather than hACE2. The protease we choose was aminopeptidase N(pepN), which is a E. coli strain K12 MG1655 protein.

As for how to detect the binding of spike protein and hACE2, we planned to apply a FRET design between the two proteins. In our design, we choosed to put both fluorescent proteins at the N terminal of spike and ACE2. Therefore, when the spike protein and hACE2 bind to each other, the acceptor of FRET pair will emit light due to the decrease of distance between the FRET pair.

Phase Two Design (Summer Vacation)

During experiment, we found various problems about our original design. First, the fusion protein with Fret(hACE2+linker+protease+fret) is far too big to construct and express in E. coli(over 5000 bp). Second, the cloning of pepN wasn’t too easy, since the genomic DNA of E. coli horbors more than 4M bp gene sequence. We had quite a hard time adjusting the PCR condition. Lastly, there were still cleavage sites on hACE2, which could lead to the inactivation of out fusion protein.

To solve these problems, we later add a his tag on the end of the plasmid to replace the usage of Fret protein. The result will be demonstrate through western blotting gel. The mass of protein various before and after protease cleavage. We also adjust the denaturing time in the three-step cycle of PCR from standard 30 second to 3 minutes, which is meant to fully open the 4M genomic DNA. Lastly, we choose to use hACE2’s RBD(receptor-binding domain) rather than full length in order to eliminate possible cleavage site on it.

What’s noteworthy is that we choose KOD as our PCR enzyme in order to prevent mutation cause by DNA polymerization. In comparision t enzyme Taq, KOD has a better proofreading accuracy and thermostability. This could provide a better quality for our engineering product. Moreover, Gidson assemble was used in order to combine multiple DNA fragments simultaneously, enhancing the efficiency of our experiment.

Phase Three Design(September ~)

In the final pahse of our experiment, we replaced pepN with pepP after analyzing the structure of pepN. This protease is structurally enclosed, making it hard to react with big protein substrate such as spike protein. PepP(aminopeptidase P) is another protease existing in E. coli strain K12 MG1655. The structure of pepP is a C-like bar, which is relatively open and likely to react to larger substrate.

According to MEROPS’s data, PepP has four cleavage sites on spike protein(326 ~ 333 IVRFPNIT, 487 ~ 494 NCYFPLQS, 1049 ~ 1056 LMSFPQSA, 1086 ~ 1093 KAHFPREG, between F&P). However, pepP left a cleavage site on hACE2’s RBD(protein sequence 364~371 DMAYAAQP, between Y&A), which could lead to further malfunction problem. As a result, we created a point mutation on hACE2, changing sequence YAA into AAA to prevent protease reaction. This design ensures the fusion protein intact and function normally.

Finally, in case pepP can’t react with full length spike protein, we created spike fragments of the cleavage sites. As research pointed out, pepP can cut small fragments of protein(roughly 4.15~4.2 kDa). Each fragment are attached with his tag. With western blotting method, we could differentiate different protein after reaction.

References

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).
Benton, D.J., Wrobel, A.G., Xu, P. et al. Receptor binding and priming of the spike protein of SARS-CoV-2 for membrane fusion. Nature (2020).
Xia, S., Liu, M., Wang, C. et al. Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion. Cell Res 30, 343–355 (2020).
https://www.ebi.ac.uk/merops/
https://www.uniprot.org/uniprot/P04825
Taq and Other Thermostable DNA Polymerases. (2008). Principles and Technical Aspects of PCR Amplification, 103–118. doi:10.1007/978-1-4020-6241-4_7
Lissandron, V., Terrin, A., Collini, M., D’alfonso, L., Chirico, G., Pantano, S., & Zaccolo, M. (2005, October 21). Improvement of a FRET-based Indicator for cAMP by Linker Design and Stabilization of Donor–Acceptor Interaction.
P. Trigo-Mourino, T. Thestrup, O. Griesbeck, C. Griesinger, S. Becker, Dynamic tuning of FRET in a green fluorescent protein biosensor. Sci. Adv. 5, eaaw4988 (2019).
Han, F., Luo, Y., Ge, N. and Xu, J. (2008), Construction of fluorescence resonance energy transfer vectors and their application in study of structure and function of signal transducers and activators of transcription 1. Acta Biochimica et Biophysica Sinica, 40: 934-942. doi:10.1111/j.1745-7270.2008.00480.x