Team:TU Darmstadt/Description
Project Description
As our lives become more and more customized by the day, so does the world of synthetic biology. In order to promote this development of individual specification, we created our own modular system: a modular Virus-like particle (MVP). Virus-like particles (VLPs) can be used for a variety of nanoscale technologies. The huge potential of VLPs has already been recognized and VLP-based technologies are in current research and development pipelines. Due to their potential of packaging substances and targeted attachment of cargo, they are used for numerous applications like vaccinations and targeted drug delivery. The potential of VLP-based technologies seems big but it reaches its limits due to the lack of a standardized method for a tailored production which could reduce production time and cost for different VLP-based applications.
Usually, when used for presenting epitopes on the exterior, Virus-like particles
(VLPs) are modified
on DNA level. Then, after expression, the VLP assembles. It consists of the VLP itself and the
desired
cargo. This approach can be difficult, if the epitope to be presented is quite big.
[1]
Besides this problem, the approach of genetic
fusion seems to be complex and not easily interchangeable. Therefore, we demonstrate an approach
where the presentation of the desired cargo on the exterior of the VLP is not achieved through
genetic fusion, but rather through an enzyme which covalently connects the cargo with the VLP.
This
approach creates the opportunity to easily modify the exterior of the VLP, and thus results in a
modular platform.
Our goal was to design a platform that everyone can modify to their personal liking in an easy and fast manner. More precisely, we decided to use the exterior of the Salmonella typhimurium P22 bacteriophage, as scientific publications have pointed out its feasibility. [2] VLPs only consist of the absolutely necessary viral capsid proteins and do not contain any viral DNA or RNA, and are consequently non-infectious. [3] For the assembly of the P22 capsid, consisting of so-called coat proteins (CP), scaffolding proteins (SP) are needed. They ensure the composition (self-assembly) of the CPs. This mechanism is navigated by the C-terminus of the scaffold protein, which interacts with the coat protein.[2] The P22 VLP is the foundation of our modular platform. Our system makes the modification of the outer surface of VLPs possible.
After synthesis of the VLP, it can be modified on the outside using an enzyme called sortase. This transpeptidase covalently binds the protein of interest to the VLP, as was shown by Patterson et al. 2017.[2] In order to accomplish the modification the coat protein is marked with a C-terminal LPETGG-tag via genetic engineering. This tag can then be recognized by the sortase and latter connects it to its counterpart protein of interest containing a N-terminal poly-glycine tag. We specifically chose two variants of the Sortase A. One of them was the Sortase A7M, a heptamutant of wildtype Sortase A, which functions calcium-independendly. This is of importance for the in vivo production of our MVPs in E. coli. Due to its calcium-independence, the Sortase A7M is not dependent on a certain Ca2+-concentration inside the cell. The particles can not only be modified to display structures on their surface, but can also contain cargo on the inside. This can either be achieved by designing a fusion protein consisting of SP and a cargo of choice [4], or by encapsulation of the desired molecule. [5]
As mentioned before, the P22 VLP is the foundation of our platform. Together with the Sortase it forms the core of "The Real MVP". For the expression system of our MVPs, we established special vectors. These contain the genetic parts of our capsids, which can be induced independently from each other. Therefore, we designed a simple bioreactor to produced VLPs in E. coli. We established and tested different purification approaches. This will enable other teams to obtain the basic VLPs. At the same time the exterior modification can be adapted according to choice.
The modification crucially depends on the sortase. This led us to investigate the efficiency of the
two Sortase A variants in a variety of different assays. In order to further improve our
modular platform, we focused our modeling on the Sortase A7M and its molecular dynamics. By
modeling the
previously unknown structure of the Sortase A7M, we gained a closer insight into the linking reaction mechanism.
We set out to develop a standardized expression system for the production of our Modular
Virus-like particles.
This will enable the iGEM community to obtain their own MVPs.
References
- ↑ Chroboczek J, Szurgot I, Szolajska E. "Virus-like particles as vaccine." Acta Biochim. Pol. 2014;61:531–539 [1]
- ↑ Patterson, D., et al., Sortase-Mediated Ligation as a Modular Approach for the Covalent Attachment of Proteins to the Exterior of the Bacteriophage P22 Virus-like Particle. Bioconjug Chem, 2017, 28(8): p. 2114-2124. [2]
- ↑ Hill, B.D., et al., Engineering Virus-like Particles for Antigen and Drug Delivery. Curr Protein Pept Sci, 2018, 19(1): p. 112-127. [3]
- ↑ O'Neil, A., et al., Genetically programmed in vivo packaging of protein cargo and its controlled release from bacteriophage P22. Angew Chem Int Ed Engl, 2011, 50(32): p. 7425-8. [4]
- ↑ Zdanowicz, M. & Chroboczek, J. "Virus-like particles as drug delivery vectors." Acta Biochim. Pol. 2016;63:469–473 [5]
