18542002095500A new COVID-19 vaccine platform depends on encapsulin protein nanoparticle
18542002095500A new COVID-19 vaccine platform depends on encapsulin protein nanoparticle
by Siriporn Maneekobkulong
Thesis submitted in fulfilment of the requirements for the degree of
Master of Science, Biomedical engineering
under the supervision of Dr. Andrew Care
00A new COVID-19 vaccine platform depends on encapsulin protein nanoparticle
by Siriporn ManeekobkulongThesis submitted in fulfilment of the requirements for the degree of
Master of Science, Biomedical engineering
under the supervision of Dr. Andrew Care
18542007124700University of Technology Sydney
Faculty of Life Science
November 2022
00University of Technology Sydney
Faculty of Life Science
November 2022
Student certification
I declare that a thesis has never been submitted for any degree and it is not being delivered as a component of a different degrees application. Secondly, I verify that I wrote it and that I acknowledge any sources involved as well as any assistance I have already gotten through its preparation.
I gave my supervisors considerable time to review many revisions of my thesis and received much criticism.
Final word count: 3,000 5,000 words
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Signature of candidate _ _____________.
Supervisor certification
Student name: _Siriporn Maneekobkulwong___
Student ID: ___14356745____
Thesis Title: A new SARS-COV-2 (COVID-19) vaccine platform depends on encapsulin protein nanoparticles.
Principal supervisor name: _Dr Andrew Care________
Principal supervisors signature: ____________
Date:______
Acknowledgements
First of all, I could not have undertaken this journey without my mentor, Dr Andrew Care, and co-mentors, . [https://www.bachelorprint.eu/thesis/acknowledgement-for-thesis/
https://www.scribbr.com/dissertation/acknowledgements/]
Abstract
Contents
Acknowledgements. .
Abstract
Abbreviations
Introduction..
Materials and Methods
Results..
Discussion.
Conclusion.
References.
Appendix..
Abbreviations
His-Tags Histidine tags
MX-Enc Myxococcus xanthus encapsulin
Qt Quasibacillus thermotolerans
QT-Enc Quasibacillus thermotolerans encapsulin
QT-ST Quasibacillus thermotolerans with C-terminal Spy tag
SARS-COV-2 Severe acute respiratory syndrome coronavirus 2
SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis
T=1 Triangulation number 1
T=3 Triangulation number 3
T=4 Triangulation number 4
TM-Enc Thermotoga maritima encapsulin
Writing 3,000 5,000 words
Reference
Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) or known as coronavirus disease pandemic of 2019 (COVID-19)
Aim and Hypothesis
The total number of COVID-19 case is 621,147,972 and the total death number is 6,556,676 in worldwide (reference WHO website).
COVID-19 variants of concern;
Alpha
Beta
Gamma
Delta
Omicron
Currently, vaccine commercial are
[Tm (T=1) Sandra (2021) the previous study showed three domains: P,E and A; A-domain is the C-terminus The studied showed the
Keywords: Nanoncage, cage protein nanoparticle, cargo, Spy Tag, His Tag, linker, nanoparticle scaffold, Escherichia coli (E. coli)
Aim of this experiment to develop a new COVID-19 vaccines platform
The hypothesis is
{edit and changing the word: encapsulins can serve as a an antigen delivery platform for the development of a plug n' play COVID-19 vaccine.}
Tm(T=1) 24 nm, Mx (T=2) and Qt (T=4)
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{**Need to editing and change because copy from article (Structural overview of encapsulin nanocompartments.
(PDB: 6NJ8). (b) Schematic of a hexameric facet (blue) and pentameric vertex (orange) capsomere assembly. (c) Schematic of icosahedron with T = 4 triangulation cage overlay (left) and the Q. thermotolerans encapsulin (PDB: 6NJ8) with the same overlay and pentameric vertices highlighted in orange (right). (d) Size comparison of a bovine serum albumin (BSA) monomer (PDB: 4F5S), the encapsulins from Thermotoga maritima (PDB: 3DKT), Myxococcus xanthus (PDB: 4PT2), Q. thermotolerans (PDB: 6NJ8), and the HK97 bacteriophage capsid (PDB: 1OHG) with pentameric vertices in orange. Figures created using ChimeraX (Goddard et al., 2018). PDB, Protein Data Bank [Color figure can be viewed at wileyonlinelibrary.com]}
Materials and Methods
Identification of suitable loop areas on encapsulin nanoparticles via computer simulation (Aim 1:identify and choosing the loop region of cargo)
For recognition of the en
Analysed the similarity of encapsulin protein cages
Encapsulin of Thermotoga maritima (Tm) (PDB code: 3DKT), Myxococcus xanthus (Mx) (PDB code: 4PT2) and Quasibacillus thermotolerans (Qt) (PDB code: A0A0F5HPP7) were used the codes from Uniprot website to get the align sequence section in FASTA file. The codes were Q9WZP2, Q1D6H4 and A0A0F5HPP7 respectively. These FASTA files were converted into the text files to use in Espript websites to compare the similarity between these encapsulins via using the PDB codes.
Recognized the flexibility of encapsulin protein nanoparticle
The sequences of individual encapsulin cage from Uniprot were inserted into MEDUSA website. Then, they were analysed to provide the different classes of the resiliences.
Determine the loop region of these encapsulin
The PDB codes were used for downloading the images of biological assembly 1 in PDB-gz file. Next, these images were uploading into ChimeraX software to analyse the loop regions that were suitable for adding Spy-Tag (AHIVMVDAYKPTK) and His-Tag (HHHHHH).
Loop segment interaction with nanoparticle is confirmed in silico
For confirmation of the inserted Spy-tag, linker and His-Tag into QT encapsulins, the four different DNA synthesiss parts of QT were made by Twist Biosciences and Integrated DNA Technology (IDT) companies. They took around 4 weeks to delivery into the laboratory to do the cell transformation, selecting the colonies, protein expression, expression evaluation, cell lysis and protein purification.
Cell Transformation
QT-cargo-loading GFP plasmid (56 ng/l) was taken 2 l
Transformation process (plasmid)
Equipment:
QT-cargo-loading GFP (super fluorescent ) plasmid tube (original stock 56 ng/ul)
NEB 5-alpha E.coli (high Efficiency) competent cell. [we use this E. Coli strains because it makes a lot of copies of plasmid while it grows and it is hard to have the mutant.]
BL media
Carbonicillin antibiotic agar plate (to select the cell culture that contains the plasmid. It is able to resist the Carbonicillin antibiotic.)
Pipetter & pipetting tips
Method:
Put the plasmid tube into the ice
Label your new tube mixed plasmid & cell and taking 2 uL of the QT-cargo-loading plasmid into the tube that contains around 100 ng/ul of the plasmid, then put it into the ice box.
Adding 5 uL of the competent cell (NEB 5-alpha) into the mixed plasmid & cell tube
Mixed it well by klicking 4-5 times with your fingers, then, put it back to the ice for 30 mins
Selecting plasmid
Protein expression
Cell expression
Transformation ->Selection-> Protein expression ->analysis of expression
Cell lysis -> Protein purification
Cell competent BL21 cell
Running gel
Results
Identify Bacteria
Tm 265 sequence length
(T=1 60 monomer assembled icosahedron composed of 60 asymmetric protein building block [Sandra, 2021]).
FASTA
Q9WZP2 [Uniprot] reference: https://www.uniprot.org/uniprot/Q9WZP2
>sp|Q9WZP2|ENCAP_THEMA Type 1 encapsulin shell protein OS=Thermotoga maritima (strain ATCC 43589 / DSM 3109 / JCM 10099 / NBRC 100826 / MSB8) OX=243274 GN=enc PE=1 SV=2
MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLGEVEVLSD
ENEVVKWGLRKSLPLIELRATFTLDLWELDNLERGKPNVDLSSLEETVRKVAEFEDEVIF
RGCEKSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINF
LKEEAGHYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKD
AVRLFITETFTFQVVNPEALILLKF
3DKT [PDB] reference: https://www.rcsb.org/structure/3DKT
>3DKT_1|Chains A, B, C, D, E, F, G, H, I, J|Maritimacin|Thermotoga maritima (2336)
MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLGEVEVLSDENEVVKWGLRKSLPLIELRATFTLDLWELDNLERGKPNVDLSSLEETVRKVAEFEDEVIFRGCEKSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGHYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETFTFQVVNPEALILLKF
Uniprot TM VS MX
Espript result of TM VS MX
TM VS QT Uniprot
TM VS QT Espript
MX VS QT Uniprot
MX VS QT Espript
Comparison between three encapsulin results from Espript
Image of the Tm loop region from ChiemiraX software
Figure :
Mx 287 {30-32 nm, made up 180 subunits (Jones JA, 2021)}
FASTA
Q1D6H4 [Uniprot] reference: https://www.uniprot.org/uniprot/Q1D6H4
>sp|Q1D6H4|ENCAP_MYXXD Type 1 encapsulin shell protein EncA OS=Myxococcus xanthus (strain DK1622) OX=246197 GN=encA PE=1 SV=2
MPDFLGHAENPLREEEWARLNETVIQVARRSLVGRRILDIYGPLGAGVQTVPYDEFQGVS
PGAVDIVGEQETAMVFTDARKFKTIPIIYKDFLLHWRDIEAARTHNMPLDVSAAAGAAAL
CAQQEDELIFYGDARLGYEGLMTANGRLTVPLGDWTSPGGGFQAIVEATRKLNEQGHFGP
YAVVLSPRLYSQLHRIYEKTGVLEIETIRQLASDGVYQSNRLRGESGVVVSTGRENMDLA
VSMDMVAAYLGASRMNHPFRVLEALLLRIKHPDAICTLEGAGATERR
4PT2 [PDB] reference: https://www.rcsb.org/sequence/4PT2
>4PT2_1|Chains A, B, C[auth P]|Encapsulin protein|Myxococcus xanthus (246197)
MPDFLGHAENPLREEEWARLNETVIQVARRSLVGRRILDIYGPLGAGVQTVPYDEFQGVSPGAVDIVGEQETAMVFTDARKFKTIPIIYKDFLLHWRDIEAARTHNMPLDVSAAAGAAALCAQQEDELIFYGDARLGYEGLMTANGRLTVPLGDWTSPGGGFQAIVEATRKLNEQGHFGPYAVVLSPRLYSQLHRIYEKTGVLEIETIRQLASDGVYQSNRLRGESGVVVSTGRENMDLAVSMDMVAAYLGASRMNHPFRVLEALLLRIKHPDAICTLEGAGATERR
Image of the Mx loop region from ChiemiraX software
Figure :
Qt {42 nm outer diameter, 240 subunits (jones Ja, 2021)}
FASTA
A0A0f5HPP7 [Uniprot] reference: https://www.uniprot.org/uniprot/A0A0F5HPP7.fasta
>sp|A0A0F5HPP7|ENCAP_QUATH Type 1 encapsulin shell protein OS=Quasibacillus thermotolerans OX=1221996 GN=enc PE=1 SV=1
MNKSQLYPDSPLTDQDFNQLDQTVIEAARRQLVGRRFIELYGPLGRGMQSVFNDIFMESH
EAKMDFQGSFDTEVESSRRVNYTIPMLYKDFVLYWRDLEQSKALDIPIDFSVAANAARDV
AFLEDQMIFHGSKEFDIPGLMNVKGRLTHLIGNWYESGNAFQDIVEARNKLLEMNHNGPY
ALVLSPELYSLLHRVHKDTNVLEIEHVRELITAGVFQSPVLKGKSGVIVNTGRNNLDLAI
SEDFETAYLGEEGMNHPFRVYETVVLRIKRPAAICTLIDPEE
6NJ8 [PDB] reference: https://www.rcsb.org/structure/6NJ8
>6NJ8_1|Chains A, B, C, D|Encapsulating protein for a DyP-type peroxidase|Quasibacillus thermotolerans (1221996)
MNKSQLYPDSPLTDQDFNQLDQTVIEAARRQLVGRRFIELYGPLGRGMQSVFNDIFMESHEAKMDFQGSFDTEVESSRRVNYTIPMLYKDFVLYWRDLEQSKALDIPIDFSVAANAARDVAFLEDQMIFHGSKEFDIPGLMNVKGRLTHLIGNWYESGNAFQDIVEARNKLLEMNHNGPYALVLSPELYSLLHRVHKDTNVLEIEHVRELITAGVFQSPVLKGKSGVIVNTGRNNLDLAISEDFETAYLGEEGMNHPFRVYETVVLRIKRPAAICTLIDPEE
>6NJ8_2|Chains E, F, G|targeting peptide|Bacillus thermotolerans (1221996)
TVGSLIQ
Image of the Qt loop region from ChiemiraX software
Figure :
After Identified each bacteria encapsulin protein nanoparticle, Qt-Enc was chosen to insert linker, which were SpyTag and HisTag
Gel Result
Figure: The image displayed the total protein expression with three temperature conditions. The red boxes showed QT-ST
The protein molecular weight band showed the combination of the Qt-Enc-spytag-HisTag
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Discussion
According to the result of the bacterial identification, Qt was chosen to do for confirming the loop region step. Because the
[Yin-Feng, 2021The SARS-CoV-2 virus relies on a spike protein on the viral membrane for hostcell recognition, attachment, and membrane fusion. The coronavirus spike protein also shares a similarity in its structural appearance as a trimeric fusion protein.7-9 As is the case in the closely related SARS-CoV, the spike protein of SARS-CoV-2 recognizes angiotensin converting enzyme 2 (ACE2) as the cell entry receptor. we used the SpyTag-SpyCatcher system to guarantee a flexible and highly efficient production of SARS-CoV-2 RBD-conjugated nanoparticles. Moreover, due to the independent expression of the RBD as the antigen and the nanoparticle scaffold, the construction and production of proteins could be achieved using different optimized expression systems.]
The advantages of this experiment are increased effective vaccine to the variants of COVID-19 spike, low cost to produce vaccine and saved money for delivery and storage of vaccine, If this experiment is successful in the clinical trail and using in the commercial.
The limitation of this experiment is short time to performance in the laboratory (Lack of time) and it might be some miss take for analyse the areas that we want to add combination of the Spy-Tag, linker and His-Tag into the encapsulins.
Future directions
For study further, if this experiment is more successful to appear the combination of the Qt cargo with the spy tag and his tag, it should go to the pre-clinical trail to test. Therefore, it can be use for COVID-19 vaccine in the future.
Conclusion
References
Andrews N, Stowe J, Kirsebom F, Toffa S, Rickeard T, Gallagher E, et al. Covid-19 Vaccine Effectiveness against the Omicron (B.1.1.529) Variant. New England Journal of Medicine. 2022;386(16):153246.
Jones JA, Giessen TW. Advances in encapsulin nanocompartment biology and engineering. Biotechnology and bioengineering. 2021;118(1):491505.
Kang Y-F, Sun C, Zhuang Z, Yuan R-Y, Zheng Q, Li J-P, et al. Rapid Development of SARS-CoV2 Spike Protein Receptor-Binding Domain Self-Assembled Nanoparticle Vaccine Candidates. ACS nano. 2021;15(2):273852.
Keeble A, Yadav V, Ferla M, Bauer C, Chuntharpursat-Bon E, Huang J et al. DogCatcher allows loop-friendly protein-protein ligation. Cell Chemical Biology. 2022;29(2):339-350.e10.
Michel-Souzy S, Hamelmann NM, Zarzuela-Pura S, Paulusse JMJ, Cornelissen JJLM. Introduction of Surface Loops as a Tool for Encapsulin Functionalization. Biomacromolecules. 2021;22(12):523442.
Sutter, M., Boehringer, D., Gutmann, S., Gunther, S., Prangishvili, D., Loessner, M.J., Stetter, K.O., Weber-Ban, E., Ban, N. (2008) Nat Struct MoI Biol 15:939-947 DOI: 10.1038/numb.1473 website link: https://www.rcsb.org/structure/3dkt
Tan TK, Rijal P, Rahikainen R, Keeble AH, Schimanski L, Hussain S, et al. A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain induces potent neutralising antibody responses. Nature communications. 2021;12(1):542542.
UCSF ChimeraX Home Page [Internet]. Cgl.ucsf.edu. 2022 [cited 18 May 2022]. Available from: https://www.cgl.ucsf.edu/chimerax/.
Vander Meersche, Y., Cretin, G., de Brevern, A. G., Gelly, J. C., & Galochkina, T. (2021). MEDUSA: Prediction of protein flexibility from sequence. Journal of Molecular Biology, 166882. https://doi.org/10.1016/j.job.2021.166882 Available website:https://www.dsimb.inserm.fr/MEDUSA/Appendix
TM result from MEDUSA
Mx result from MEDUSA
Qt result from MEDUSA
FASTA sequences of proteins produced in this study
From Uniprot
>Tm
-MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLGEVE---
-----VLSDENEVVKWG-LRKSLPLIELRATFTLDLWELDNLERGKPNVDLSSLEETVRK
VAEFEDEVIFRGCEKSGVKGLLSFEERKIE---CGSTPKDLLEAIVRALSIFSKDGIEGP
YTLVINTDRWINFLKEEAGHYPLEKRVEECLRGGKIITTPRI--EDALVVSERGGDFKLI
LGQDLSIGYEDREKDAVRLFITETFTFQVVNPEALILLKF-------
>Mx
MPDFLGHAENPLREEEWARLNETVIQVARRSLVGRRILDIYGPLGAGVQTVPYDEFQGVS
PGAVDIVGEQETAMVFTDARKFKTIPIIYKDFLLHWRDIEAARTHNMPLDVSAAAGAAAL
CAQQEDELIFYGDARLGYEGLMTANGRLTVPLGDWTSPGGGFQAIVEATRKLNEQGHFGP
YAVVLSPRLYSQLHRIYEKTGVLEIETIRQLASDGVYQSNRLRGESGVVVSTGRENMDLA
VSMDMVAAYLGASRMNHPFRVLEALLLRIKHPDAICTLEGAGATERR
>Qt
MNKSQLYPDSPLTDQDFNQLDQTVIEAARRQLVGRRFIELYGPLGRGMQSVFNDIFMESH
EAKMDFQGSFDTEVE-SSRRVNYTIPMLYKDFVLYWRDLEQSKALDIPIDFSVAANAARD
VAFLEDQMIFHGSKEFDIPGLMNVKGRLTHLIGNWYESGNAFQDIVEARNKLLEMNHNGP
YALVLSPELYSLLHRVHKDTNVLEIEHVRELITAGVFQSPVLKGKSGVIVNTGRNNLDLA
ISEDFETAYLGEEGMNHPFRVYETVVLRIKRPAAICTLIDPEE----
