Although the initial attempts to investigate the interaction between the bat rotavirus protein G3p[10] and the LNDF1 substrate did not yield successful binding, the modification of the G3p[10] protein by adding and subsequently cleaving the GST tag presents a promising track for future experimentation. Moving forward, we plan to further explore the effects of the GST tag on the G3p[10] protein’s ability to interact with LNDF1. Specifically, we will assess the efficiency and stability of the enzyme-substrate complex post-GST tag cleavage to determine if this modification significantly enhances binding.
In addition, we will continue to refine the experimental conditions for crystal formation that may improve the interaction between the G3p[10] protein and LNDF1. These modifications may provide valuable insights into the structural and functional dynamics of the VP8-glycan interaction.
Future Directions:
Conducting additional assays to quantitatively measure the binding affinity between the Spike G3p[10] protein and LNDF1 in the presence and absence of the GST tag.
Investigating potential structural changes in the G3p[10] protein after GST tag cleavage using techniques such as X-ray crystallography.
We also could consider exploring alternative glycan substrates to determine if G3p[10] shows more efficient binding to other glycan molecules, which may offer broader implications for understanding the role of G3p[10] in rotavirus infection
NACE Competencies
1. Critical Thinking and Problem Solving
Our research required troubleshooting challenges like low protein yield and unsuccessful substrate binding during crystallization. Addressing these issues, such as transitioning from His-tag to GST-tag purification methods, helped strengthened our ability to assess problems, analyze alternatives, and seek out new strategies.
2. Communication
Effectively communicating our research findings, both written and verbally, has been a vital part of this experience. From presenting experimental results to creating clear, organized research posters and websites, we have learned to translate detailed scientific concepts into accessible formats for diverse audiences.
3. Professionalism and Work Ethic
Working in a research lab demands reliability, attention to detail, and responsibility. Balancing independent tasks, collaboration, and meeting research deadlines helped us develop strong work habits, time management skills, and a commitment to our work.
References:
International Committee on Taxonomy of Viruses (ICTV). (2023). Rotavirus. In Virus Taxonomy: 2023 Release. Retrieved from https://ictv.global/report/chapter/sedoreoviridae/sedoreoviridae/rotavirus
Thermo Fisher Scientific. (n.d.). Overview of Affinity Purification. Retrieved from https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-affinity-purification.html
Dóró, R., Martella, V., Leshem, E., & Gentsch, J.R. (2015). Emerging Rotavirus Strains: A Review of Global Trends and Zoonotic Potential. Infection, Genetics and Evolution, 34, 302-312. https://doi.org/10.1016/j.meegid.2015.06.002
→ Reviews global trends in zoonotic RV strains like G3P[10] and discusses cross-species transmission risks.
Hu, L., et al. (2012). Cell Attachment Protein VP8 of a Rotavirus Strain Implicated in Neonatal Diarrhea Binds to Type I and Type II Histoblood Group Antigens*. Journal of Virology, 86(22), 13059-13067. https://doi.org/10.1128/JVI.01176-12
→ Describes how VP8* interacts with host cell surface molecules, essential for viral attachment and host specificity.